Method of analyzing multiple samples simultaneously by detecting absorption and systems for use in such a method

ABSTRACT

The present invention provides a method of analyzing multiple samples simultaneously by absorption detection. The method comprises:  
     (i) providing a planar array of multiple containers, each of which contains a sample comprising at least one absorbing species,  
     (ii) irradiating the planar array of multiple containers with a light source comprising or consisting essentially of at least one wavelength of light that is absorbed by one or more of the absorbing species, the absorption of which is to be detected, and  
     (iii) detecting absorption of light by one or more of the absorbing species with a detection means that is in line with the light source and is positioned in line with and parallel to the planar array of multiple containers at a distance of at least about 10 times a cross-sectional distance of a container in the planar array of multiple containers measured orthogonally to the plane of the planar array of multiple containers. The detection of absorption of light by a sample in the planar array of multiple containers indicates the presence of an absorbing species in the sample. The method can further comprise:  
     (iv) measuring the amount of absorption of light detected in (iii) for an absorbing species in a sample. The measurement of the amount of absorption of light detected in (iii) indicates the amount of the absorbing species in the sample.  
     Also provided by the present invention is a system for use in the above method. The system comprises:  
     (i) a light source comprising or consisting essentially of at least one wavelength of light that is absorbed by one or more absorbing species, the absorption of which is to be detected,  
     (ii) a planar array of multiple containers, into each of which can be placed a sample comprising at least one absorbing species, and  
     (iii) a detection means that is in line with the light source and is positioned in line with and parallel to the planar array of multiple containers at a distance of at least about 10 times a cross-sectional distance of a container in the planar array of multiple containers measured orthogonally to the plane of the planar array of multiple containers.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application is a continuation of copendingapplication Ser. No. 10/070,531, filed Jun. 5, 2002, which is the U.S.national phase of International Patent Application No. PCT/US00/20447,filed Jul. 28, 2000, which claims the benefit of U.S. Provisional PatentApplication No. 60/153,263, filed Sep. 9, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under ContractNo. W-7405-Eng-82 awarded by the U.S. Department of Energy. Therefore,the Government may have certain rights to this invention.

FIELD OF THE INVENTION

[0003] This invention relates to a method of analyzing multiple samplessimultaneously by detecting absorption and systems for use in such amethod.

BACKGROUND OF THE INVENTION

[0004] The rapid development of biological and pharmaceutical technologyhas posed a challenge for high-throughput analytical methods. Forexample, current development of combinatorial chemistry has made itpossible to synthesize hundreds or even thousands of compounds per dayin one batch. Characterization and analysis of such huge numbers ofcompounds have become the bottleneck. Parallel processing (i.e.,simultaneous multi-sample analysis) is a natural way to increase thethroughput. However, due to limitations related to column size, pressurerequirements, detector and stationary phase material, it will be verydifficult to build a highly multiplexed high-performance liquidchromatography (HPLC) system. The same goes for building a highlymultiplexed gas chromatography (GC) system.

[0005] High performance capillary electrophoresis (CE) has rapidlybecome an important analytical tool for the separation of a largevariety of compounds, ranging from small inorganic ions to largebiological molecules. With attractive features such as rapid analysistime, high separation efficiency, small sample size, and low solventconsumption, CE is being increasingly used as an alternative orcomplementary technique to HPLC. For example, the use of capillary gelelectrophoresis has greatly improved DNA sequencing rates compared toconventional slab gel electrophoresis. Part of the improvement in speed,however, has been offset by the loss of the ability (inherent in slabgels) to accommodate multiple lanes in a single run. Highly multiplexedcapillary electrophoresis, by making possible hundreds or even thousandsof parallel sequencing runs, represents an attractive approach toovercoming the current throughput limitations of existing DNA sequencinginstrumentation. Such a system has been disclosed in U.S. Pat. Nos.5,582,705 (Yeung et al.), 5,695,626 (Yeung et al.), and 5,741,411 (Yeunget al.). In this system, light-induced fluorescence is exclusivelyemployed as the detection method.

[0006] While fluorescence detection is suitable for DNA sequencingapplications because of its high sensitivity and special labelingprotocols, UV absorption detection has remained very useful because ofits ease of implementation and wide applicability, especially for thedeep-UV (200-220 nm) detection of organic and biologically importantcompounds. A capillary isoelectric focusing system using atwo-dimensional CCD detector, in which one dimension represents thecapillary length and the other dimension records the absorptionspectrum, has been described by Wu and Pawliszyn, Analyst (Cambridge),120, 1567-1571 (1995). The system has been used for two capillary tubesbut is not easily adapted for three or more capillary tubes because thesystem requires the capillary tubes to be separated by space. Instead ofproviding wavelength resolution in the second CCD dimension, isoelectricfocusing in two capillary tubes is simultaneously monitored. The use ofoptical fibers for illumination, however, has led to low lightintensities and poor UV transmission. So, only visible wavelengths havebeen employed for the detection of certain proteins. Because the CCD hasa very small electron well capacity (about 0.3 million electrons), thelimit of detection (LOD) of this system is limited by the high shotnoise in absorption detection. The use of the CCD produces anoverwhelming amount of data per exposure, limiting the data rate to oneframe every 15 seconds. Also, the imaging scheme utilized is notsuitable for densely packed capillary arrays because of the presence ofmechanical slits to restrict the light paths. Further, in order to avoidcross-talk, only square capillaries can be used.

[0007] Photodiode arrays (PDA) are used in many commercial CE and HPLCsystems for providing absorption spectra of the analytes in real time.Transmitted light from a single point in the flow stream is dispersed bya grating and recorded across the linear array. A capillary zoneelectrophoresis system using a photodiode array as the imagingabsorption detector has been described by Culbertson and Jorgenson,Anal. Chem., 70, 2629-2638 (1998). Different elements in the array areused to image different axial locations in one capillary tube to followthe progress of the separation. Because the PDA has a much largerelectron well capacity (tens of million electrons), it is superior tothe CCD for absorption detection. Time-correlated integration is appliedto improve the signal-to-noise ratio (S/N).

[0008] What is still needed is an absorption detection approach for thesimultaneous analysis of multiple systems. One such system is shown inU.S. Pat. No. 5,900,934 (Gilby et al.). This system includes aphotodetector array comprising a plurality of photosensitive elementsconnected to provide a serial output. The elements are typically pixelsof a photodiode array (PDA). The elements are illuminated by a lightsource positioned to illuminate at least a portion of the photodetectorarray. The light source may be an AC or DC mercury lamp or other useablelight source for chromatography. An array of separation channels isdisposed between the light source and the photodetector array, each ofthe separation channels having a lumen, a sample introduction end and adetection region disposed opposite the sample introduction end. Thearray is a multiple parallel capillary electrophoresis system. A maskelement having at least one aperture for each associated separationchannel is required. Each aperture corresponds to its associatedseparation channel, thereby selectively permitting light from the lightsource to pass through the lumen of its associated separation channel.At least a portion of the light passing through the lumen of theassociated separation channel falls on a respective photosensitiveelement of the photodetector array to effect measurement of absorptionof light by a sample introduced into the sample introduction end of theassociated separation channel.

[0009] The system described by Gilby et al. has disadvantages because itlimits the amount of light impinging on the separation channel,providing less than desirable light intensity to the PDA. Further,aligning the apertures and the mask elements with the separationchannels, e.g., capillaries, is difficult for several reasons. Forexample, positioning the capillaries with equal separation there betweenis difficult as the capillaries generally are not of equal dimension,e.g., diameter tolerances vary greatly. Further, for example, the maskgeometry does not provide identical light paths, which leads tononlinear response. Also, a mask can produce stray light, which leads topoor detection limits, and does not completely eliminate crosstalk fromthe adjacent capillaries, since the light beams are diverging and cannotescape the detector element. In addition, a mask can be difficult tomanufacture, due to the requirement of uniformity. Also, Gilby placesthe sample and the PDA too close together, resulting in stray light,cross talk and the inability to use the maximum pathlength of light.

[0010] Thus, in view of the disadvantages inherent to the methods andsystems in the art, there remains a need a method of analyzing multiplesamples simultaneously by absorption detection. It is an object of thepresent invention to provide such a method. It is another object of thepresent invention to provide a system for use in such a method. Theseand other objects and advantages of the present invention as well asadditional inventive features will become apparent to one of ordinaryskill in the art from the detailed description provided herein.

[0011] The present invention also addresses other disadvantages in theart. For example, since the invention of the polymerase chain reaction(PCR) in 1985 by Kary Mullis, the ultimate in sensitivity, together withincreasing ease in implementation, have placed this technique in acentral position in molecular biology research and in clinical diagnosis(Rolf et al., PCR: Clinical Diagnostics and Research, 1992,Springer-Verlag, Berlin and Heidelberg). In the last ten years, PCR hasstimulated numerous investigations in genetic analysis, and is evenbeing used to determine the genetic basis of complex diseases (Sacki etal., Science 230: 1350 (1985)). There is no need to reiterate thedevelopment of CE as a powerful analytical tool in post-PCR analysis. Alarge amount of research has been done to explore the advantages of CEover traditional slab gel electrophoresis, including high-speed,high-resolution restriction fragments analysis (Guttman et al., Anal.Chem. 62: 2348 (1992); Milofsky et al., Anal. Chem. 65: 153 (1993);Williams et al., J. Chromatogr. A680: 525 (1994); Chang et al., J.Chromatogr. B669: 113 (1995); Barron et al., Electrophoresis 16: 64(1995); and Righetti et al., Anal. Biochem. 244: 195 (1997)),high-speed, high-throughput DNA sequencing (Ruiz-Martinez et al., Anal.Chem. 65: 2851 (1993); Lu et al., J. Chromatogr. A680: 497 (1994); Lu etal., J. Chromatogr. A680: 503 (1994); Fung et al., Anal. Chem. 67: 1913(1995); Zhang et al., Anal. Chem. 67: 4589 (1995); Carrilho et al.,Anal. Chem. 68: 3305 (1996); and Kim et al., J. Chromatogr. 781: 315(1997)), rapid and precise DNA typing and sizing (Baba et al.,Electrophoresis 16: 1437 (1995); Noble, Anal. Chem. 67: 613A (1995);Zhang et al., Anal. Chem. 68: 2927 (1996); Isenberg et al.,Electrophoresis 17: 1505 (1996); Zhang et al., J. Chromatogr. A768: 135(1997); Butler et al., Electrophoresis 16: 974 (1995); and Wang et al.,Anal. Chem. 67: 1197 (1995)), single-base mutation analysis (Marino etal., Electrophoresis 17: 1499 (1996); Arakawa et al., J. Chromatogr.:A664: 89 (1994); Hebenbrock et al., Electrophoresis 16: 1429 (1995);Kuypers et al., J. Chromatogr.: B 675: 205 (1996); Cheng et al., J. Cap.Elec. 2: 24 (1995); and Ren et al., Anal. Biochem. 245: 9 (1997)) andthe analysis of disease causing genes (Lu et al., Nature 368: 269(1994); Felmlee et al., J. Cap. Elec. 2: 125 (1995); Gelfi et al.,BioTechniques 19: 254 (1995); and Grossman et al., Nucleic Acids Res.22: 4527 (1994)). In particular, capillary array electrophoresis, alongwith other micro-fabricated devices (Ueno et al., Anal. Chem. 66: 1424(1994); Takahashi et al., Anal. Chem. 66: 1021 (1994); and Anazawa etal., Anal. Chem. 68: 2699 (1996)) are promising methods for the purposeof achieving high-throughput DNA analysis. In this regard, singlecapillaries have been utilized for DNA analysis (Guttman et al. (1992),supra).

[0012] The conventional protocol for DNA analysis calls for labelingwith radionuclides or fluorescent tags before, during or aftersize-based separation in slab gel electrophoresis or in capillary gelelectrophoresis (CGE). This derivatization process involves expensivereagents and raises safety concerns for the operator and for wastedisposal because of the toxic nature of these labeling reagents.

[0013] The present invention can be applied to genetic typing anddiagnosis based simply on UV absorption detection. The additivecontribution of each base pair to the total absorption signal providesadequate detection sensitivity for analyzing most PCR products. Not onlyis the use of specialized and potentially toxic fluorescent labelseliminated, but also the complexity and cost of the instrumentation aregreatly reduced. The DNA analysis protocols can, therefore, be designedto take advantage of high-throughput capillary array gel electrophoresisand simple UV absorption detection, based on the inherent spectralproperties of the DNA bases. UV absorption detection of DNA productsreduces the cost of analysis, since it does not require labeling.

[0014] Similarly, peptide mapping represents one of the most powerfuland successful tools available for the characterization of proteins(Garnick et al., Anal. Chem. 60: 2546-2557 (1988); Borman, Anal. Chem.59: 969A-973A (1987)). Although less informative than proteinsequencing, it allows rapid analysis with simple instrumentation. Inpeptide mapping, a sample protein is selectively cleaved by enzymes orby chemical digestion (Tarr et al., Anal. Biochem. 131: 99-107 (1983);Dong, Advances in Chromatography 32: 22-51, Marcel Dekker, Inc.: NewYork (1992); Geisow et al., Biochem. J. 161: 619-625 (1977); and Ward etal., J. Chromatogr. 519: 199-216 (1990)). The peptide map then serves asa unique fingerprint of the protein and can accurately reveal verysubtle differences among individual variants. Trypsin is by far the mostwidely used proteolytic enzyme in peptide mapping. Its desirablefeatures are that cleavage at the C-terminal side of lysine and arginineis generally quantitative under proper conditions and that trypsintolerates concentrations of urea as high as 4 M (Dong (1992), supra).The disadvantage is that the fragments formed may be too small,averaging 7-12 amino acid residues, resulting in very complex trypticmaps. After tryptic digestion, the digest is typically analyzed byvarious methods, such as slab gel electrophoresis (Cleveland et al., J.Biol. Chem. 252: 1102-1106 (1977)), thin-layer chromatography (TLC)(Stephens, Anal. Biochem. 84: 116-126 (1978)), HPLC (Hancock et al.,Anal. Biochem. 89: 203-212 (1978); Cox et al., Anal. Biochem. 154:345-352 (1986); Fullmer et al., J. Biol. Chem. 254: 7208-7212 (1979);Vensel et al., J. Chromatogr. 266: 491-500 (1983); Leadbeater et al., J.Chromatogr. 397: 435-443 (1987); Dong et al., J. Chromatogr. 499:125-139 (1990); and Hartman et al., J. Chromatogr. 360: 385-395 (1986),and capillary zone electrophoresis (CZE) (Jorgenson et al., J. HighResolut.Chromatogr. Commun. 4: 230-231 (1981); Cobb et al., Anal. Chem.61: 2226-2231 (1989); Chang et al., Anal. Chem. 65: 2947-2951 (1993);Nashabeh et al., J. Chromatogr. 536: 31-42 (1991); Ward et al., J.Chromatogr. 519: 199-216 (1990); Janini et al., J. Chromatogr. 848:417-433 (1999); Frenz et al., J. Chromatogr. 480: 379-391 (1989); andGrossman et al., Anal. Chem. 61: 1186-1194 (1989)) to yield a peptidemap. Gradient reversed-phase HPLC is the most common form of peptidemapping in use today (Leadbeater et al. (1987), supra; Dong et al.,(1990), supra; and Hartman et al. (1986), supra).

[0015] In particular, CZE has received considerable attention as acomplementary method to reversed-phase liquid chromatography in peptidemapping efforts (Jorgenson et al. (1981), supra; Cobb et al. (1989),supra; Chang et al. (1993), supra; Nashabeh et al. (1991), supra; Wardet al. (1990), supra; Janini et al. (1999), supra; Frenz et al. (1989),supra; and Grossman et al. (1989), supra). Separation of variouspeptides can be optimized through pH adjustments. Through the additionof micelle-forming surfactants to the running buffer, a dynamicpartition mechanism (i.e., hydrophobicity) of peptide separation canalso be established for the neutral fragments. Although CE is quiteefficient and fast for analyzing peptide fragments, the completeseparation of peptides in a digest of high molecular mass proteins, forexample, is not possible by using a single buffer condition. UnlikeHPLC, the implementation of gradient separation in CE is not trivial(Whang et al., Anal. Chem. 64: 502-506 (1992); and Chang et al., J.Chromatogr. B 608: 65-72 (1992)).

[0016] Although these methods are useful for characterizing proteins,there are still other problems, such as the relatively large amount ofsample required, long analysis time, and efficiency of thederivatization reaction. Also, a typical map contains 20-150 peaks, allof which should ideally be totally resolved (Dong et al. (1992), supra).Therefore, a high degree of column resolution and system precision arerequired to reproduce accurately the maps, preferably starting withsubnanomolar quantities.

[0017] The present invention enables a peptide map to be obtained thatcan serve as a unique fingerprint of the protein. Reliablehigh-throughput analyses can be performed, for example, based onmulti-dimensional CE and a single prescribed experimental protocol.

[0018] Combinatorial screening also has attracted much attentionrecently because of its ability efficiently and reliably to zero in andidentify the best solution to a chemical or biochemical question(Borman, C&E News, Mar. 8, 1999, pages 33-60). In chemical synthesis,optimization of the reaction yield can be achieved by simultaneouslyexploring all possible reaction conditions, catalysts and reagents. Indrug discovery, all related structural variants of a given candidate canbe tested against the target. However, screening must be comprehensiveso that there is no chance of missing the best combination. Thisdictates having a large number of experiments to cover many parametersand to extend the range of each of these parameters. High throughput isa requirement in order to produce a timely result. It is primarilybecause of the advances in high-throughput technologies and automationthat combinatorial screening became practical. Still needed are generaland rugged analytical methodologies that can keep up with the largenumber of reactions that can be performed in any given time. Anotherissue is miniaturization of the entire operation. This impacts the costof reagents, proper disposal of solvents, space for manipulation andstorage, etc.

[0019] Currently, there are several parallel assays for screeninghomogeneous catalysts. Modifications in UV absorption (Wagner et al.,Sicence 270: 1797-1800 (1995); Menger et al., J. Org. Chem. 63:7578-7579 (1998)), fluorescence (Cooper et al., J. Am. Chem. Soc. 120:9971-9972 (1998); Shaughnessy et al., J. Am. Chem. Soc. 121: 2123-2132(1999)), color (Lavastre et al., Chem. Int. Ed. 38: 3163-3165 (1999)) ortemperature (Taylor et al., Science 280: 267-270 (1998); Reetz et al.,Angew. Chem. Int. Ed. 37: 2647-2650 (1999)) induced by the catalyticreactions are indicators of catalytic activity. In these approaches,although the relative activity of the catalyst is determined quickly, noquantitative information about the overall yield or the regioselectivityand stereoselectivity of the process can be obtained. It is alsonecessary that the product exhibit very different measurable propertiescompared to the solvent or the reagents. Most of the time, secondaryscreening is necessary. Mass spectrometry (MS) (Orschel et al., Angew.Chem. Int. Ed. 38: 2791-2794 (1999)), which also has been widely used toscreen catalysts, can provide selective detection. However, to addressstereoselectivity, these procedures still tend to be laborious (Reetz etal., Angew. Chem. Int. Ed. 38: 1758-1761 (1999)). So far, MS is still aserial, rather than a parallel, approach, although the analysis time isreasonably short.

[0020] Separation-based techniques can solve the above problems. Serialmethods, which include HPLC and CE, have been used to analyze asymmetriccatalysis (Porte et al., J. Am. Chem. Soc. 120: 9180-9187 (1998); Dinget al., Angew. Chem. Int. Ed. 38: 497-501 (1999)) and alkylationreactions (Gaus et al., Biotech. & Bioeng. 1998/1999 61: 169-177). Thethroughput that can be achieved with serial separation schemes is loweven with special techniques, such as sequential sample injection (Rocheet al., Anal. Chem. 69: 99-104 (1997)) and sample multiplexing (Woodburyet al., Anal. Chem. 67: 885-890 (1995)). Multiplex HPLC is anotherinteresting approach (Gong et al., Anal. Chem. 71: 4989-4996 (1999)),but achieving a high degree of multiplexing, such as 96 capillaries incapillary array electrophoresis (CAE), is not trivial. Thin-layerchromatography and gel electrophoresis, on the other hand, are difficultto completely automate.

[0021] A highly successful format for combinatorial screening is that ofDNA chips (Southern, Electrophoresis 16: 1539-1542 (1995); Chee et al.,Science 274: 610-614 (1996); and Winzeler et al., Science 281: 1194-1197(1998)). A comprehensive set of oligonucleotides immobilized within asmall area is used to identify specific target sequences byhybridization. Oligonucleotide chips also have been used to developaptamers that exhibit specific protein-nucleotide binding (Weiss et al.,J. Virol. 71: 8790-8797 (1997)). Such heterogeneous screening assayshave benefited from sensitive detection based on laser-inducedfluorescence (LIF), either by selective labeling or by selectivequenching. For homogeneous assays, the 96-well microtiter plate is apopular format. Fluidic operations, plate readers and autosamplers tointerface to standard analytical instruments have been developed forthis format. When there is a color (absorption) change or fluorescencechange, detection and quantitation is straightforward. In manysituations, however, the reaction mixture is complex and some degree ofseparation or purification is needed before measurement. Multiple liquidchromatographs or single instruments with several columns can inprinciple be used for analysis of the reaction mixtures. Still, muchhigher throughput and much smaller sample sizes, which means muchsmaller amounts of reagents, are desirable. The present inventionenables such higher throughput and smaller sample sizes and does notrequire the species of interest to be fluorescent.

BRIEF SUMMARY OF THE INVENTION

[0022] The present invention provides a method of analyzing multiplesamples simultaneously by absorption detection. The method comprises:

[0023] (i) providing a planar array of multiple containers, each ofwhich contains a sample comprising at least one absorbing species,

[0024] (ii) irradiating the planar array of multiple containers with alight source comprising or consisting essentially of at least onewavelength of light that is absorbed by one or more of the absorbingspecies, the absorption of which is to be measured, and

[0025] (iii) detecting absorption of light by one or more of theabsorbing species with a detection means that is in line with the lightsource and is positioned in line with and parallel to the planar arrayof multiple containers at a distance of at least about 10 times across-sectional distance of a container in the planar array of multiplecontainers measured orthogonally to the plane of the planar array ofmultiple containers. The detection of absorbtion of light by a sample inthe planar array of multiple containers indicates the presence of anabsorbing species in the sample. The method can further comprise:

[0026] (iv) measuring the amount of absorption of light detected in(iii) for an absorbing species in a sample. The measurement of theamount of absorption of light detected in (iii) indicates the amount ofthe absorbing species in the sample.

[0027] Also provided by the present invention is a system for use in theabove method. The system comprises:

[0028] (i) a light source comprising or consisting essentially of atleast one wavelength of light that is absorbed by one or more absorbingspecies, the absorption of which is to be detected,

[0029] (ii) a planar array of multiple containers, into each of whichcan be placed a sample comprising at least one absorbing species, and

[0030] (iii) a detection means that is in line with the light source andis positioned in line with and parallel to the planar array of multiplecontainers at a distance of at least about 10 times a cross-sectionaldistance of a container in the planar array of multiple containersmeasured orthogonally to the plane of the planar array of multiplecontainers.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 is a diagram of a system for use in the present inventivemethod.

[0032]FIG. 2A is a graph of counts vs. pixel number.

[0033]FIG. 2B is a graph of counts vs. pixel number.

[0034]FIG. 3 is a graph of absorbance vs. frame number.

[0035]FIG. 4 is a graph of light intensity (counts) vs. frame number vs.value after subtraction (counts).

[0036]FIG. 5 is a graph of intensity vs. frame number.

[0037]FIG. 6 is the result of CZE separation of four visible dyes in a96 capillary array.

[0038]FIG. 7 is a graph of migration time vs. capillary number.

[0039]FIG. 8 is a graph of peak area vs. capillary number.

[0040]FIG. 9 is a reconstructed two-dimensional electropherogram forcapillary array electrophoresis.

[0041]FIG. 10 is a set of extracted electropherograms for capillaryarray electrophoresis.

[0042]FIG. 11 represents the peptide maps of three variants of bovineβ-lactoglobulin [SEQ ID NO: 1].

[0043]FIG. 12 shows the results of the six-dimensional separations(capillary vs. migration time) of tryptic digests of BLGA and BLGB inthe 96-capillary array.

[0044]FIG. 13 shows selected electropherograms of BLGA extracted fromthe data in FIG. 12.

[0045]FIG. 14 shows selected electropherograms of BLGB extracted fromthe data in FIG. 12.

[0046] FIGS. 15A-D show typical peptide maps of BLGA and BLGB at four pHconditions (FIG. 15A at pH 9.3; FIG. 15B at pH 8.1; FIG. 15C at pH 5.0;and FIG. 15D at pH 2.5) for CZE using a single capillary after trypticdigestion.

[0047]FIG. 16 shows MEKC peptide maps of BLGA (B and D) and BLGB (A andC) obtained at two different MEKC conditions using a nonionic surfactant(A and B) and/or the combination of nonionic and anionic surfactant (Cand D).

[0048]FIG. 17 shows the effect of Tween 20 concentration at pH 8.1 onthe MEKC peptide maps for BLGB in terms of migration time (min).

[0049]FIG. 18A is a graph of the ratio of the amount of NADH (injected)to the amount of NAD (injected) vs. the results from nine electrokineticinjections.

[0050]FIG. 18B is a graph of the ratio of the amount of NADH (injected)to the amount of NAD (injected) vs. the results from hydrodynamic nineinjections.

[0051]FIG. 19 is a reconstructed absorption image of combinatorialscreening of enzyme activity in a 96 capillary array in which thecapillaries (1-96) are displayed from top to bottom and migration time(0-33 min) is plotted from left to right.

[0052]FIG. 20 is an electropherogram of products after 180 min reactionfor different LDH concentrations at pH=7.

[0053]FIG. 21 is an electropherogram of products after 180 min reactionfor different pH at an LDH concentration of 5×10⁻⁹ M.

[0054]FIG. 22 is a graph of NADH conversion percentage vs. pH for series1-9 at 180 min incubation.

[0055]FIG. 23 is a graph of reaction percentage vs. pH for series 1-9for 30 min of LDH catalysis.

[0056]FIG. 24 which is a graph of reaction percentage vs. pH value forseries 1-9 for 24 hr of LDH catalysis.

[0057]FIG. 25 shows the separation of two isomeric forms (A and B) ofthe product from the reagents and the internal standard using twodifferent buffers (1a and 1 b).

[0058]FIG. 26 shows a 96 capillary separation for the reactionconditions for 1 b in FIG. 25 and a hydrodynamic injection of 1 min, inwhich the horizontal direction spans 88 capillaries (the remaining 8capillaries contained solvent only and were not plotted) and thevertical direction represents time.

[0059]FIG. 27 is a 3-dimensional bar graph of yield vs. catalyst vs.base.

[0060]FIG. 28 is a selectivity plot of two isomers produced in thereactions, wherein P1/P2 is the ratio of the two isomers A and B,respectively.

[0061]FIG. 29 is a line graph of fractional conversion vs. time (hr) vs.base, for the reaction using Pd(PPh₃)₄ as the catalyst and variousbases.

DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention provides a method of analyzing multiplesamples simultaneously by absorption detection. The present inventionutilizes an integrated approach toward achieving automation, high speed,high accuracy and low cost, such as in the context of multiplexedelectrophoresis. The method can be used, for example, in multicapillaryarray zone electrophoresis, micellar electrokinetic chromatography,capillary electrochromatography, and capillary gel electrophoresis. Whena multicapillary array is used, as much as 100 times, or even 1,000times or greater, higher analysis throughput can be achieved whencompared to conventional single-capillary electrophoresis. The system isat least about 100-fold more sensitive than the system of Wu andPawliszyn.

[0063] The method comprises:

[0064] (i) providing a planar array of multiple containers, each ofwhich contains a sample comprising at least one absorbing species,

[0065] (ii) irradiating the planar array of multiple containers with alight source comprising or consisting essentially of at least onewavelength of light that is absorbed by one or more of the absorbingspecies, the absorption of which is to be detected, and

[0066] (iii) detecting absorption of light by the one or more absorbingspecies with a detection means that is in line with the light source andis positioned in line with and parallel to the planar array of multiplecontainers at a distance such that stray light exiting the planar arrayof multiple containers disperses prior to impinging upon the detectionmeans. The amount of stray light falls off inversely as the square ofthe distance between the planar array and the detector increase. Thus,the light impinging upon the detection means is substantially only thatwhich is transmitted through the multiple containers. In this manner,the intensity of the outputs from the planar array of multiplecontainers is the strongest and, therefore, the intensity of the outputsfrom the detection means is also the strongest, thereby making thedetermination of the intensity outputs from the detection means for eachcontainer in the planar array of multiple containers easy. The detectionof absorption of light by a sample in the planar array of multiplecontainers indicates the presence of an absorbing species in the sample.

[0067] The method can further comprise (iv) measuring the amount ofabsorption of light detected in (iii) for an absorbing species in asample. The measurement of the amount of absorption of light detected in(iii) indicates the amount of the absorbing species in the sample.Methods of measuring the amount of absorption of light are known in theart. Basically, one measures the intensity of light in the absence andpresence of a sample. The logarithm of the ratio is the absorbance(Beer-Lambert law). Preferably, the distance between the planar array ofmultiple containers and the detection means is at least about 10 times,at which distance the stray light is less than about 1%, morepreferably, at least about 100 times, a cross-sectional distance of acontainer in the planar array of multiple containers measuredorthogonally to the plane of the planar array of multiple containers.

[0068] The distance between the light source and the planar array ofmultiple containers is not critical to the practice of the presentinvention. However, the shorter the distance between the light sourceand the planar array of multiple containers, the more light will bereceived by the planar array of multiple containers. The greater thedistance between the light source and the planar array of multiplecontainers, the more uniform will be the light received by the planararray of multiple containers. The more light that the planar array ofmultiple containers receives, the more sensitive will be the detection.

[0069] The position of the light source in relation to the planar arrayof multiple containers also is not critical to the practice of thepresent invention as long as the light source irradiates the planararray of multiple containers. Other considerations are as noted in thepreceding paragraph.

[0070] Preferably, the distance between the planar array of multiplecontainers and the detection means is at least about 10 times, morepreferably, at least about 100 times, a cross-sectional distance of acontainer in the planar array of multiple containers measuredorthogonally to the plane of the planar array of multiple containers.Thus, the distance between the planar array of multiple containers andthe detection means is preferably from about 1 cm to about 30 cm, morepreferably from about 3 cm to about 30 cm, and most preferably fromabout 10 cm to about 30 cm. When cylindrical capillary tubes are used asthe multiple containers, preferably the distance is from about 1 cm toabout 30 cm, more preferably from about 3 cm to about 30 cm, and mostpreferably from about 10 cm to about 30 cm.

[0071] By “multiple containers” is meant at least three or more,preferably at least about 10, more preferably at least about 90, anddesirably as many as can be accommodated by the system described herein.While the multiple containers can comprise any suitable containers,desirably the multiple containers allow the passage of light from thelight source through the walls of the containers facing the lightsource, through the samples in the containers, and through the walls ofthe containers facing the detection means. Thus, the walls of thecontainers are desirably transparent, although, in some instances, thewalls of the containers can be translucent. In this regard, it is notnecessary for the entirety of the walls of the containers to allow thepassage of light from the light source as described above as long as atleast a portion of the walls of the containers allow the passage oflight from the light source such that the samples in the containers areirradiated and the light that is not absorbed by the absorbing speciesin the samples is detectable by the detection means. Preferably, themultiple containers comprise cylindrical capillary tubes. Preferably,the planar array of multiple containers comprises at least about 10capillary tubes, more preferably at least about 90 capillary tubes, suchas 96 capillary tubes, and desirably as many as can be accommodated bythe system described herein.

[0072] The planar array desirably further comprises at least one controlcontainer. However, if the light source is stable, a control containeris not necessary.

[0073] In general, the containers used in the planar array should havesmooth surfaces and uniformly thick walls and be made of a material thatis transparent over the range of wavelengths of light absorbed by anabsorbing species in a sample, the absorbance of which is to be detectedor measured. Preferred materials for containers include, but are notlimited to, plastics, quartz, fused silica (in particular for capillarytubes) and glass. The cross-section of a container is not critical tothe present inventive method. However, the smaller the cross section ofthe container, the more useful is the container in highly multiplexedapplications as a greater number of containers can be used in a smalleramount of space. Similarly, the thickness of the wall of the containeris not critical to the present inventive method. The wall should be ofsufficient thickness so as to maintain the structural integrity of thecontainer, yet not so thick as to impede adversely the passage of lightthrough the container. The shape of the container also is not criticalto the present inventive method. The container can have any suitableshape. Desirably, the shape of the container is conducive to beingclosely packed and minimizes the generation of stray light by thecontainer.

[0074] A cylindrical capillary tube is a preferred container for use inthe context of the present invention. Capillary tubes are commerciallyavailable from a number of sources, including Polymicro Technologies,Inc., Phoenix, Ariz. The capillary tube is preferably coated with apolymer, such as polyimide, so that it is mechanically stable. Thecoating must be removed in the region to be irradiated by the lightsource. An excimer laser can be used to remove the polymer coating.

[0075] Preferably, the multiple containers in the planar array arearranged substantially parallel to each other. Also preferably, themultiple containers in the planar array are also arranged substantiallyadjacent to each other. For example, when the multiple containers arecapillary tubes, the capillary tubes are closely packed so as to besubstantially contiguous along their parallel lengths, leavingessentially no space between adjacent capillaries. Substantiallyadjacent capillary tubes can be physically touching each other along allor a portion of their lengths, although slight inconsistencies incapillary wall diameter or other features of the array can prevent themfrom being in contact along their entire lengths. The planar arraydesirably is rigidly mounted to reduce flicker noise.

[0076] If a large amount of heat is generated during the method,particularly in the vicinity of the planar array of multiple containers,cooling should be employed to dissipate the heat. Excessive heat canlead to mechanical vibrations between adjacent containers in the planararray of multiple containers (e.g., such as in the case of closelypacked capillary tubes), which, in turn, can lead to excess noise. Alaminar flow of nitrogen gas, such as in parallel to the portion of thecontainers undergoing detection, can be used for cooling.

[0077] The detection means can comprise any suitable means of detectingabsorption. Preferably, the detection means comprises a plurality ofabsorption detection elements, such as a plurality of photosensitiveelements, which desirably are positioned in a linear array, although atwo-dimensional image array detector can be used. Desirably, thedetection means is parallel to and in line with the linear array ofmultiple containers. The detection means desirably is rigidly mounted toreduce flicker noise. In this regard, the relative positions of cellcomponents used in the system must be fixed.

[0078] Preferably, a linear photodiode array (PDA) is used. Desirably,the PDA incorporates a linear image sensor chip, a driver/amplifiercircuit and a temperature controller, which desirably thermoelectricallycools the sensor chip to a temperature from about 0° C. to about −40° C.Lowering the temperature lowers the dark count and minimizes thetemperature drift, thus enabling reliable measurements to be made over awide dynamic range. The driver/amplifier circuit is desirably interfacedto a computer via an I/O board, which preferably also serves as a pulsegenerator to provide a master clock pulse and a master start pulse,which are required by the linear image sensor. The PDA records the imagelinearly—not two-dimensionally. Preferably, the data acquired arewritten directly to the hard disk in real time. Also, preferably, thesignals from up to at least about ten elements of the PDA are displayedin real time.

[0079] Alternatively, a charge-coupled device (CCD) or acharge-injection device (CID) can be used. However, the CCD records intwo dimensions, which is less efficient, requiring more computer memory,is slower, requires every location to be read (not a single line likePDA). Furthermore, whereas a CCD has only 100,000 electrons in eachlocation, each element in a PDA can store 59 million electrons per pixelper location; thus, given that detection sensitivity is related to thesquare root of the number of electrons that can be detected, a PDA isorders of magnitude more sensitive than a CCD.

[0080] Preferably, the PDA comprises linearly aligned pixels, in whichcase each container in the planar array of multiple containers desirablyis a capillary tube and each capillary tube preferably is opticallycoupled to less than about ten pixels, more preferably from about 7 toabout 9 pixels, some of which are coupled to the walls of the capillaryand some of which are coupled to any space between the walls of adjacentcapillaries and at least one of which is coupled to the lumen of thecapillary. Thus, the stray light caused by the walls of a capillary isdispersed prior to striking the pixels and/or is confined to the pixelscoupled to the side walls and generally does not affect the signalproduced by the pixel coupled to the lumen of the capillary. While theratio of capillaries to optically coupled pixels is preferably less thanabout 1:10, more preferably from about 1:7 to about 1:9, the ratio ofcapillaries to optically coupled pixels need not be an integer ratio.

[0081] If the detection means is a PDA comprising linearly alignedpixels, step (iii) of the method can comprise selecting one pixel fromthe middle group of pixels, i.e., the pixel detecting the strongestlight intensity and using that pixel to detect the absorbance by thetarget species. When more than one pixel is optically coupled to theinterior of a container, it is desirable to select only one to analyzeto and to disregard the others. Alternatively, only one pixel can beoptically coupled to each container, obviating the need to make a pixelselection, although this is less preferred because of the need forcritical optical alignment. In large arrays with many containers, whichdesirably are capillary tubes, it can be practical to use a higher ratioof pixels to containers so as to accommodate inconsistencies andvariations in packing of the containers, and the width of the walls ofthe containers; etc. Where higher ratios of pixels to capillaries areused, more than one pixel can be optically coupled to the lumen of acontainer. Each pixel that is coupled to the lumen of a container willproduce a signal having an intensity directly proportional to theintensity of light detected. The pixel producing the signal having thegreatest intensity, i.e., the “brightest” pixel, is advantageouslyselected.

[0082] Selection of the appropriate pixel from those that are opticallycoupled to the interior portion of a container can be conveniently doneby way of a calibration step. Thus, the method of the present inventioncan further comprise a calibration step, which is performed prior tointroducing the samples into the containers. Alternatively, every nthcapillary, e.g., every 10th capillary, includes a control or blanksample, i.e., a control container as indicated above.

[0083] The method can be carried out at ambient temperature, such asroom temperature, such as from about 20° C. to about 30° C., or as lowas 0° C. or as high as 80° C. However, if the method employs a PDA asthe detection means as is preferred, desirably the PDA has its owncooler for operation at subzero temperatures, such as from about 0° C.to about −40° C.

[0084] The light source preferably comprises or consists essentially ofa wavelength in the range from about 180 nm to about 1500 nm. Examplesof suitable light sources include mercury (for ultraviolet (UV) lightabsorption), tungsten (for visible light absorption), iodine (for UVlight absorption), zinc (for UV light absorption), cadmium (for UV lightabsorption), xenon (for UV light absorption), deuterium (for visiblelight absorption), and the like. Desirably, the light source comprisesor consists essentially of a wavelength of light that will be absorbedby an absorbing species, the absorption of which is to be detected.Which wavelength of light will be absorbed by an absorbing species ofinterest, i.e., an absorbing species, the absorption of which is to bedetected or measured in accordance with the present invention, can bedetermined using a standard absorption spectrometer. Alternatively,spectroscopic tables that provide such information are available in theart, such as through the National Institute of Science and Technology(NIST). Desirably, a maximally absorbed wavelength of light is selectedfor a given absorbing species to be detected or measured such thatsmaller amounts of the absorbing species can be detected. Generally, thelight source provides light impinging on the planar array of multiplecontainers orthogonal to the plane in which the planar array of multiplecontainers. The light source can be a point source. Also preferably, thelight source has a power output of about 0.5 mW to about 50 mW. Thelight source can be AC or DC, although DC is preferred. Any flickernoise from the light source can be eliminated by using a double beam oflight.

[0085] The pathlength of light is critical to the sensitivity of thepresent inventive method. The longer the pathlength of light absorbed bya sample in a container, the larger the signal for the sample. This isin accordance with Beer's Law, which states that absorbance=constant(which is the spectral characteristic of an absorbing species in asample in a container, the absorbance of which is to be detected ormeasured)×pathlength of the light×concentration of the absorbing speciesin a sample in a container. A high constant and a long pathlength aredesired.

[0086] An optical filter desirably is positioned between the planararray of multiple containers and the detection means. The optical filterselects at least one wavelength of light from the light source that isabsorbed by an absorbing species, the absorption of which is to bedetected. While an optical filter can be positioned between the lightsource and the planar array of multiple containers in addition to, or asan alternative to, an optical filter positioned between the planar arrayof multiple containers and the detection means, the placement of asingle optical filter between the light source and the planar array ofmultiple containers is disadvantageous inasmuch as it does not block thesubsequent fluorescence by the sample from reaching the detection means.In contrast, the placement of an optical filter between the planar arrayof multiple containers and the detection means blocks samplefluorescence from reaching the detection means.

[0087] A flat-field lens also desirably is positioned between the planararray of multiple containers and the detection means. The flat-fieldlens couples light that is not absorbed by the one or more absorbingspecies in each sample in the planar array of multiple containers withthe detection means. While a lens that is not a flat-field lens can beused in the context of the present invention, it is disadvantageousinasmuch as it does not image the entire field evenly. Consequently, theedges of the field are distorted and the absorption of the containers inthe planar array of multiple containers positioned at the edges of thefield of the lens cannot be detected or measured. The lens inverts theimage of the planar array of multiple containers onto the face of thedetection means, which preferably is a PDA.

[0088] Desirably, the coupling of light by the flat-field lens isshielded from the light source. This way, only the light from the lensis focused on the detection means.

[0089] The detection limit of rhodamine 6G for each capillary in aplanar array of multiple capillaries is about 1.8×10⁻⁸ M. The cross-talkbetween adjacent capillaries in a planar array of multiple capillariesis less than about 0.2%.

[0090] While the sample can be introduced into each capillary tube in aplanar array of multiple capillaries by any suitable method, preferablythe sample is introduced into the capillary tube by pressure, gravity,vacuum, capillary or electrophoretic action.

[0091] A beam expander can be positioned between the light source andthe planar array of multiple containers. The beam expander can alter thefocused line of the light source so as to irradiate more effectively themultiple containers. The beam, optionally, can be altered or redirected,as with a mirror, filter or lens, prior to contacting the array.

[0092] A collimating focusing lens can be positioned between the lightsource and the planar array of multiple containers.

[0093] The above components are placed to eliminate substantially and,desirably, completely, stray light. There are two kinds of stray light.One kind of stray light is the glare that results from the containershaving side walls and interior lumens. The other kind of stray light isthat which is due to the presence of other containers in the planaryarray of multiple containers. This kind of stray light is referred to as“cross talk.” Cross talk essentially is the glare from other containers.Thus, there needs to be sufficient distance between the sample and theflat-field lens to eliminate substantially and, desirably completely,the two kinds of glare. A distance of at least about 1/r², i.e., therate of decrease of stray light as the distance r increases, or 1/d, inwhich r=radius and d=diameter, will eliminate most of the glare from thecontainers. Glare can be assessed by measuring a totally absorbingmaterial in a container; if there is any light that is detected, thatlight is due to glare.

[0094] Preferably, raw data sets are extracted into single-diodeelectropherograms and analyzed by converting the transmitted lightintensities collected at the PDA to absorbance values. Root-mean-squarednoise in the electropherograms is obtained using a section of baselinenear one of the analyte peaks. A preferred manner of collecting andanalyzing data obtained in accordance with the present invention is setforth in Example 1.

[0095] Mathematical smoothing can be used to reduce the noisesignificantly, without distorting the signal. See, for example,Example 1. In this regard, as high a data acquisition rate as possibleshould be employed to provide more data points for smoothing. Boxcarsmoothing, such as 25 point boxcar smoothing, is a preferred method ofmathematical smoothing.

[0096] In view of the above, the present invention further provides asystem for use in the above method, preferred embodiments of which areexemplified in the Examples and FIG. 1 set forth herein. The systemcomprises:

[0097] (i) a light source comprising or consisting essentially of atleast one wavelength of light that is absorbed by one or more absorbingspecies, the absorption of which is to be detected,

[0098] (ii) a planar array of multiple containers, into each of whichcan be placed a sample comprising at least one absorbing species, and

[0099] (iii) a detection means that is in line with the light source andis positioned in line with and parallel to the planar array of multiplecontainers at a distance such that stray light exiting the planar arrayof multiple containers disperses prior to impinging upon the detectionmeans. Thus, the light impinging upon the detection means issubstantially only that which is transmitted through the multiplecontainers. In this manner, the intensity of the outputs from the planararray of multiple containers is the strongest and, therefore, theintensity of the outputs from the detection means is also the strongest,thereby making the determination of the intensity outputs from thedetection means for each container in the planar array of multiplecontainers easy. Preferably, the distance is at least about 10 times,more preferably, at least about 100 times, a cross-sectional distance ofa container in the planar array of multiple containers measuredorthogonally to the plane of the planar array of multiple containers.The detection of absorption of light by a sample in the planar array ofmultiple containers indicates the presence of an absorbing species inthe sample.

[0100] As indicated above, the distance between the light source and theplanar array of multiple containers is not critical to the practice ofthe present invention. However, the shorter the distance between thelight source and the planar array of multiple containers, the more lightwill be received by the planar array of multiple containers. The greaterthe distance between the light source and the planar array of multiplecontainers, the more uniform will be the light received by the planararray of multiple containers. The more light that the planar array ofmultiple containers receives, the more sensitive will be the detection.

[0101] The position of the light source in relation to the planar arrayof multiple containers also is not critical to the practice of thepresent invention as long as the light source irradiates the planararray of multiple containers. Other considerations are as noted in thepreceding paragraph.

[0102] Preferably, the distance between the planar array of multiplecontainers and the detection means is at least about 10 times, morepreferably, at least about 100 times, a cross-sectional distance of acontainer in the planar array of multiple containers measuredorthogonally to the plane of the planar array of multiple containers.Thus, the distance between the planar array of multiple containers andthe detection means is preferably from about 1 cm to about 30 cm, morepreferably from about 3 cm to about 30 cm, and most preferably fromabout 10 cm to about 30 cm. When capillary tubes are used as themultiple containers, preferably the distance is from about 1 cm to about30 cm, more preferably from about 3 cm to about 30 cm, and mostpreferably from about 10 cm to about 30 cm.

[0103] By “multiple containers” is meant at least three or more,preferably at least about 10, more preferably at least about 90, anddesirably as many as can be accommodated by the system described herein.While the multiple containers can comprise any suitable containers,desirably the multiple containers allow the passage of light from thelight source through the walls of the containers facing the lightsource, through the samples in the containers, and through the walls ofthe containers facing the detection means. Thus, the walls of thecontainers are desirably transparent, although, in some instances, thewalls of the containers can be translucent. In this regard, it is notnecessary for the entirety of the walls of the containers to allow thepassage of light from the light source as described above as long as atleast a portion of the walls of the containers allow the passage oflight from the light source such that the samples in the containers areirradiated and the light that is not absorbed by the absorbing speciesin the samples is detectable by the detection means. Preferably, themultiple containers comprises cylindrical capillary tubes. Ifcylindrical capillary tubes are used, preferably the distance betweenthe detection means and the planar array of multiple containers is atleast about 10 times, more preferably at least about 100 times, thediameter of a capillary tube. Preferably, the planar array of multiplecontainers comprises at least about 10 cylindrical capillary tubes, morepreferably at least about 90 cylindrical capillary tubes, such as 96cylindrical capillary tubes, and desirably as many as can beaccommodated by the system described herein.

[0104] The planar array desirably further comprises at least one controlcontainer. However, if the light source is stable, a control containeris not necessary.

[0105] In general, the containers used in the planar array should havesmooth surfaces, uniformly thick walls, and be made of a material thatis transparent over the range of wavelengths of light absorbed by anabsorbing species in a sample, the absorbance of which is to be detectedor measured. Preferred materials for containers include, but are notlimited to, quartz, fused silica (in particular for capillary tubes) andglass. The cross-section of a container is not critical to the presentinventive method. However, the smaller the cross section of thecontainer, the more useful is the container in highly multiplexedapplications as a greater number of containers can be used in a smalleramount of space. Similarly, the thickness of the wall of the containeris not critical to the present inventive method. The wall should be ofsufficient thickness so as to maintain the structural integrity of thecontainer, yet not so thick as to impede adversely the passage of lightthrough the container. The shape of the container also is not criticalto the present inventive method. The container can have any suitableshape. Desirably, the shape of the container is conducive to beingclosely packed and minimizes the generation of stray light by thecontainer.

[0106] A capillary tube is a preferred container for use in the contextof the present invention. Capillary tubes are commercially availablefrom a number of sources, including Polymicro Technologies, Inc. Thecapillary tube is preferably coated with a polymer, such as polyimide,so that it is mechanically stable. The coating must be removed in theregion to be irradiated by the light source. An excimer laser can beused to remove the polymer coating.

[0107] Preferably, the multiple containers in the planar array arearranged substantially parallel to each other. Also preferably, themultiple containers in the planar array are also arranged substantiallyadjacent to each other. For example, when the multiple containers arecapillary tubes, the capillary tubes are closely packed so as to besubstantially contiguous along their parallel lengths, leavingessentially no space between adjacent capillaries. Substantiallyadjacent capillary tubes can be physically touching each other along allor a portion of their lengths, although slight inconsistencies incapillary wall diameter or other features of the array can prevent themfrom being in contact along their entire lengths. The planar arraydesirably is rigidly mounted to reduce flicker noise.

[0108] If a large amount of heat is generated during use of the system,particularly in the vicinity of the planar array of multiple containers,the system desirably further comprises a cooling means. Excessive heatcan lead to mechanical vibrations between adjacent containers in theplanar array of multiple containers (e.g., such as in the case ofclosely packed capillary tubes), which, in turn, can lead to excessnoise. A laminar flow of nitrogen gas, such as in parallel to theportion of the containers undergoing detection, can be used.

[0109] The detection means can comprise any suitable means of detectingabsorption. Preferably, the detection means comprises a plurality ofabsorption detection elements, such as a plurality of photosensitiveelements, which desirably are positioned in a linear array, although atwo-dimensional image array detector can be used. Desirably, thedetection means is parallel to and in line with the linear array ofmultiple containers. The detection means desirably is rigidly mounted toreduce flicker noise.

[0110] Preferably, a linear photodiode array (PDA) is used. Desirably,the PDA incorporates a linear image sensor chip, a driver/amplifiercircuit and a temperature controller, which desirably thermoelectricallycools the sensor chip to a temperature from about 0° C. to about −40° C.Lowering the temperature lowers the dark count and minimizes thetemperature drift, thus enabling reliable measurements to be made over awide dynamic range. The driver/amplifier circuit is desirably interfacedto a computer via an I/O board, which preferably also serves as a pulsegenerator to provide a master clock pulse and a master start pulse,which are required by the linear image sensor. The PDA records the imagelinearly—not two-dimensionally. Preferably, the data acquired arewritten directly to the hard disk in real time. Also, preferably, thesignals from up to at least about ten elements of the PDA are displayedin real time.

[0111] Alternatively, a charge-coupled device (CCD) or acharge-injection device (CID) can be used. However, the CCD records intwo dimensions, which is less efficient, requiring more computer memory,is slower, requires every location to be read (not a single line likePDA), and has a reduced electron capacity. Furthermore, whereas a CCDhas only 100,000 electrons in each location, each element in a PDA canstore 59 million electrons per pixel per location; thus, given thatdetection sensitivity is related to the square root of the number ofelectrons that can be detected, a PDA is orders of magnitude moresensitive than a CCD.

[0112] Preferably, the PDA comprises linearly aligned pixels, in whichcase each container in the planar array of multiple containers desirablyis a capillary tube and each capillary tube preferably is opticallycoupled to less than about ten pixels, more preferably from about 7 toabout 9 pixels, some of which are coupled to the walls of the capillaryand some of which are coupled to any space between the walls of adjacentcapillaries and at least one of which is coupled to the lumen of thecapillary. Thus, the stray light caused by the walls of a capillary isdispersed prior to striking the pixels and/or is confined to the pixelscoupled to the side walls and generally does not affect the signalproduced by the pixel coupled to the lumen of the capillary. While theratio of capillaries to optically coupled pixels is preferably less thanabout 1:10, more preferably from about 1:7 to about 1:9, the ratio ofcapillaries to optically coupled pixels need not be an integer ratio.Optical coupling of the capillaries and the pixels in this mannerrenders the system extremely stable.

[0113] Given that noise will ultimately determine the minimum baselinefluctuation level and, thus, the limit of detection (LOD) of the system,as explained in Example 1, it is desirable to have as high a photoncount as possible. Preferably, at least about 300,000, more preferably,at least about 3 million, and most preferably, at least about 30 millionphotons are used. In addition, so as to allow for baseline drift due touncontrollable variables over the period of data acquisition, the diodespreferably are only 85-90% saturated.

[0114] The light source preferably comprises or consists essentially ofa wavelength in the range from about 180 nm to about 1500 nm. Examplesof suitable light sources include mercury, tungsten, iodine, zinc,cadmium, xenon, deuterium, and the like. Desirably, the light sourcecomprises or consists essentially of a wavelength of light that will beabsorbed by an absorbing species, the absorption of which is to bedetected. Which wavelength of light will be absorbed by an absorbingspecies of interest, i.e., an absorbing species, the absorption of whichis to be detected or measured in accordance with the present invention,can be determined using a standard absorption spectrometer.Alternatively, spectroscopic tables that provide such information areavailable in the art, such as through NIST. Desirably, a maximallyabsorbed wavelength of light is selected for a given absorbing speciesto be detected or measured such that smaller amounts of the absorbingspecies can be detected. Generally, the light source provides lightimpinging on the planar array of multiple containers orthogonal to theplane in which the planar array of multiple containers. The light sourcecan be a point source. Also preferably, the light source has a poweroutput of about 0.5 mW to about 50 mW. The light source can be AC or DC,although DC is preferred. Any flicker noise from the light source can beeliminated by using a double beam of light.

[0115] Desirably, an optical filter is positioned between the planararray of multiple containers and the detection means. The optical filterselects at least one wavelength of light from the light source that isabsorbed by an absorbing species, the absorption of which is to bedetected. While an optical filter can be positioned between the lightsource and the planar array of multiple containers in addition to, or asan alternative to, an optical filter positioned between the planar arrayof multiple containers and the detection means, the placement of asingle optical filter between the light source and the planar array ofmultiple containers is disadvantageous inasmuch as it does not block thesubsequent fluorescence by the sample from reaching the detection means.In contrast, the placement of an optical filter between the planar arrayof multiple containers and the detection means blocks samplefluorescence from reaching the detection means.

[0116] Also desirably, a flat-field lens is positioned between theplanar array of multiple containers and the detection means. Theflat-field lens couples light that is not absorbed by the one or moreabsorbing species in each sample in the planar array of multiplecontainers with the detection means. While a lens that is not aflat-field lens can be used in the context of the present invention, itis disadvantageous inasmuch as it does not image the entire fieldevenly. Consequently, the edges of the field are distorted and theabsorption of the containers in the planar array of multiple containerspositioned at the edges of the field of the lens cannot be detected ormeasured. The lens inverts the image of the planar array of multiplecontainers onto the face of the detection means, which preferably is aPDA.

[0117] Preferably, the system further comprises a shield that shieldsthe coupling of light by the flat-field lens from the light source. Thisway, only the light from the lens is focused on the detection means.

[0118] The detection limit for rhodamine 6G for each capillary in aplanar array of capillary tubes in the system is about 1.8×10⁻⁸ M. Thecross-talk between adjacent capillaries is less than about 0.2%.

[0119] If the system utilizes capillary tubes or the like, the systemfurther comprises a means to introduce the sample into the capillarytube. Preferably, the sample is introduced into the capillary tube bypressure, gravity, vacuum, capillary or electrophoretic action.

[0120] The system can further comprise a beam expander between the lightsource and the planar array of multiple containers. The beam expandercan alter the focused line of the light source so as to irradiate moreeffectively the multiple containers. The beam, optionally, can bealtered or redirected, as with a mirror, filter or lens, prior tocontacting the array.

[0121] The system can further comprise a collimating focusing lensbetween the light source and the planar array of multiple containers.

[0122] The above components are placed to eliminate substantially and,desirably completely, stray light as described above. Thus, there needsto be sufficient distance between the sample and the flat-field lens toeliminate substantially and, desirably completely, the two kinds ofglare. A distance of at least about 1/r² or 1/d, in which r=radius andd=diameter, will eliminate most of the glare from the containers.

[0123] Desirably, the above components are collectively placed in alight-tight construct, such as a metal box attached to an optical table.Also, desirably, the components are centered above the optical table.

EXAMPLES

[0124] The present invention is further demonstrated by way of thefollowing examples, which serve to illustrate the present invention butare not intended to limit its scope in any way.

[0125] Fluorescein (F), rhodamine 6G, 5(6)-carboxyfluorescein (5CF,6CF), β-lactoglobulin A and B (BLGA and BLGB),L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin,CHES (2-[N-cyclohexylamino]ethanesulfonic acid), tricine(N-tris[hydroxymethyl]methylglycine), Trizma®·Base(tris[hydroxymethyl]aminomethane), ammonium acetate, CaCl2,poly(vinylpyrrolidone) (PVP), sodium pyruvate (99+%), β-nicotinamineadenine dinucleotide, reduced form (β-NADH), L-lactate dehydrogenase(LDH-5(M4) 98+% isoenzyme suspension in 2.1 M (NH4)2SO4), and sodiumdodecyl sulfate (SDS) were purchased from Sigma Chemical Co. (St. Louis,Mo.). Both of the solutions of β-NAD+ and β-NADH were freshly preparedand kept in a refrigerator before the experiment. The NADH solution wascovered by black tape to prevent exposure to light. Tween 20 waspurchased from Aldrich Chemical Co. (Milwaukee, Wis.).2,7-diacetate,dichloro-fluorescein (DADCF) was obtained from Acros (FairLawn, N.J.). Ethidium bromide (EtBr) was obtained from Molecular Probes,Inc. (Eugene, Oreg.). 50-bp and 100-bp DNA ladders were purchased fromLife Technologies (Gaithersburg, Md.). The sample solutions for CZE wereprepared by dissolving the appropriate amounts of these fluoresceins in1×TBE (0.089 M Tris, 0.089 M borate, and 0.002 M ethylene diaminetetraacetic acid (EDTA) in water) buffer with 0.2% (w/w) PVP. For theMEKC experiments, the analytes and buffer additives were purchased fromAldrich (Milwaukee, Wis.), J. T. Baker (Phillipsburg, N.J.) and SigmaChemical Co. The running buffer was prepared by adding appropriatealiquots of 1.0 M HCl, 250 mM Brij-S stock solutions, acetonitrile and2-propanol into water. The pH was adjusted to 2.4 using 0.1 M HCl or 0.1M NaOH stock solution and confirmed by a pH meter. 1×TBE buffer wasprepared by dissolving pre-mixed TBE buffer powder (Amresco, Solon,Ohio) in deionized water. The coating matrix used in Example 2 was madeby dissolving 2% (w/v) of 1,300,000 MW PVP into the buffer, shaking for2 min and letting it stand for 1 h to get rid of bubbles. Poly(ethyleneoxide) (PEO) was obtained from Aldrich Chemical (Milwaukee, Wis.). Thesieving matrix used in Example 2 was made by dissolving 2% (w/v) 600,000MW PEO into the buffer. The solution was stirred vigorously overnightuntil all the material was dissolved and no bubbles could be observed.All buffers for Example 3 were filtered through a Corning® FilterSystem, 0.22-μm cellulose acetate filters (Corning, N.Y.) or μStar LB™,0.22-μm cellulose acetate non-pyrogenic filters (Coaster, Cambridge),and degassed prior to use. The water used to prepare solutions inExample 3 was deionized with a Milli-Q water purification system(Millipore, Worcester, Mass.). Bacteria-free 0.2 ml 96-well preloadedplates were obtained from Marsh Biomedical Products, Inc. (Rochester,N.Y.). Sodium phosphate monobasic (NaH2PO4.H2O) was purchased fromFisher (Fair Lawn, N.J.). All water used in Example 4 was purified by aMillipore water purification system to make sure that there was noenzyme contamination.

Example 1

[0126] This example demonstrates a multiplexed capillary electrophoresissystem that employs a single linear photodiode array detector.

[0127] Ninety six fused-silica capillaries (75 μm i.d., 150 μm o.d.;Polymicro Technologies, Phoenix, Ariz.) with 35 cm effective length and55 cm total length were packed side by side. An excimer laser beam wasused to burn off the polyimide coating in the same region of eachcapillary to provide a “window” for passage of light from a light sourcethrough a sample to be introduced into and contained within eachcapillary. At the ground, i.e., exit, end of the capillary array, thecapillaries were bundled together to allow simultaneous buffer fillingand rinsing. At the injection end, the capillary array was spread outand mounted onto a copper plate to form an 8×12 format with dimensionsthat fit into a 96-well microtiter plate for sample introduction. Inaddition, 96 gold-coated pins (Mill-Max Mfg. Corp., Oyster Bay, N.Y.)were located next to the capillary tips to serve as individualelectrodes. Samples and buffer trays were moved and aligned under thecapillary inlets. This way, the capillary array was never physicallymoved. A high-voltage DC power supply from Spellman (Plainview, N.Y.)provided power for electrophoresis. All 96 electrodes were connected tothe same power supply.

[0128] A light source, an optical, i.e., interference, filter, acapillary array holder, a camera lens and a PDA detector were placed ina light-tight metal box attached to an optical table. All opticalcomponents were centered 12.6 cm above the optical table. As the lightsource, a 12-V tungsten lamp or a 254 nm hand-held mercury lamp (modelE-09816-02; Cole-Parner, Vernon Hills, Ill.) was used for visible orultraviolet absorption detection, respectively. A diagram of a systemfor use in the present inventive method is shown in FIG. 1. In the caseof the tungsten lamp (with a filament length of 1.1 cm), the light wasfirst expanded through a cylindrical lens to cover uniformly the“windows” of the entire array of capillary tubes, which had a combinedwidth of 1.5 cm. The hand-held mercury lamp proved to have a long enoughemission length (7 cm), thus no beam expander was needed forilluminating the entire array. The transmitted light from the capillaryarray passed through an interference filter (Oriel, Stamford, Conn.) anda quartz lens (Nikon, Melville, N.Y.; f.l.=105 mm; F#=4.5). Theinterference filter was employed to define the absorption wavelength. Aninverted image of the capillary array, at a nominal magnification factorof 1.5, was created by the quartz lens on the face of the PDA. The PDA(model S5964, Hamamatsu, Bridgewater, N.J.) incorporated a linear imagesensor chip, a driver/amplifier circuit and a temperature controller.The linear image sensor chip had 1,024 dodes, each of which was 25 μm inwidth and 2,500 μm in height. The temperature controllerthermoelectrically cooled the sensor chip to 0° C. to lower the darkcount and to minimize temperature drift, thus enabling reliablemeasurements to be made over a wide dynamic range. The built-indriver/amplifier circuit was interfaced to an IBM-compatible computer(233 MHz Pentium, Packard Bell) via a National Instrument PCI E seriesmultifunction 16-bit I/O board. The I/O board also served as a pulsegenerator to provide a master clock pulse and a master start pulserequired by the linear image sensor. All codes used to operate the PDAand to acquire the data were written in-house using National InstrumentsLabview 4.1 software (Austin, Tex.). The distance between the planedefined by the capillary array and the plane defined by the PDA detectorelements was 30 cm.

[0129] A very large amount of data were generated for each CE run usingthe 1,024-element PDA detector. A run of one hour with a dataacquisition rate of 10 Hz generated 70 Mb of data. All the data were,therefore, written directly to the hard disk in real time. Signals fromup to ten elements of the PDA could be displayed in real time in theLabview program. Real-time monitoring of more pixels was limited by thevideo speed of our computer. The raw data sets were extracted intosingle-diode electropherogram data by another in-house Labview program.Data treatment and analysis were performed using Microsoft Excel 97(Microsoft, Seattle, Wash.) and GRAMS/32 5.05 (Galactic Industries,Salem, N.H.). The transmitted light intensities collected at the PDAwere converted to absorbance values using the tenth capillary (buffersolution only) as a continuous blank reference, i.e., a control. Theroot-mean-squared (rms) noise in all of the electropherograms wasobtained using a section of baseline near one of the analyte peaks. Thisbaseline section was of about the same width as the peaks of interest.

[0130] For the capillary zone electrophoresis experiments, the capillaryarray was first flushed with methanol and then water for clean up.Buffer (pH 8.0, 1×TBE with 0.2% (w/w) polyvinylpyrrolidone (PVP)) wasfilled into the capillary array while the injection end was immersedinto a buffer tray. After buffer filling, the filling ends were immersedinto a second buffer tray. The analytes were put into a 96-wellmicrotiter sample plate (1 μl/well) and injected at the cathode for 6sec at 11 kV (100 V/cm). The running voltage was also 11 kV. For the CEexperiments, the capillaries were washed for 1 min with buffer betweenruns. For the MEKC experiments, the capillary array was first flushedwith 0.1 M HCl and then water. The buffer additive used was Brij-S madeby sulfonation of Brij-30 with chlorosulfonic acid (Ding et al., Anal.Chem. 70: 1859-1865 (1998)). The analytes were injected at the cathodefor 3 sec at 10 kV and run at the same voltage.

[0131] A typical 96-capillary array image obtained using a tungsten lampand the PDA is shown in FIG. 2A, which is a graph of counts vs. pixelnumber and represents the image on the PDA of the entire 96-capillaryarray. As can be seen in FIG. 2B, which is a graph of counts vs. pixelnumber and represents the image on the PDA of one region of the96-capillary array, the center of each capillary corresponds to a ‘peak’(a center peak represented by (a)) in the image. Between two adjacentcapillaries, there is normally a spacing that also creates atransmission ‘peak’ (a spacing peak represented by (b)). These ‘spacingpeaks’ are usually a bit broader and have larger intensities (saturatedin this case) than the ‘center peaks’ in this imaging system, as shownin FIG. 2B. If the array packing is not even, two adjacent capillariescan overlap each other so that the ‘spacing peak’ between them is notobserved. Including the spacing, each capillary was imaged upon 9-11diodes in the PDA. The 96-capillary array covered 912 pixels in total.As can be seen in FIG. 2B, between the ‘center peak’ and the ‘spacingpeak,’ there is a ‘valley’ (represented by (c)), which corresponds tothe wall of the capillary. When the capillary array image waswell-focused onto the PDA, the intensities of these valleys becameminimized. This feature was used to produce the best focusing. Thediodes that corresponded to the center peaks were used as the absorptiondetectors for the corresponding capillaries. Their intensities are about40% to 90% of the saturation value of the diodes. To maintain therelatively uniform intensity distribution over the capillary array, wefound that the emission length of the light source should be at leasttwo times larger than the width of the capillary array (1.5 cm). Thehand-held mercury lamp (7-cm emission length) that was used as the UVlight source was long enough for uniform illumination of the entirearray. The tungsten lamp used as the visible light source, however, hadonly a 1-cm emission length, so a cylindrical lens needed to be added tomagnify the light source to meet the illumination requirement. FIG. 2Ashows a 2× variation in optical throughput from the center to the edgeof the array. This means that the detection limit will vary by {squareroot}{square root over (2)}X across the array. The sensitivity (signal),however, will vary by 2× unless the intensities are first ratioed to theblank (buffer) and a log scale is used (Beer's Law). Given that themercury lamp was placed very far from the array, the intensitydistribution was, thus, much more uniform than that in FIG. 2A.

[0132] No mechanical slits were used to define the image. While thecylindrical capillaries do refract light onto other diodes in the array,the distance from the array to the camera lens was maintained at adistance that was greater than the radius of curvature. Each diodereceives a low level of stray light averaged over all capillaries. Thissets the limit on the “valleys” in FIG. 2 but contributes negligibly tocross-talk. The rays of light crossing the diameters of the capillarieswill travel straight and are properly imaged at the PDA. Sensitivity(absorption path length) is, thus, also optimized. Given that there areno mechanical slits and each capillary spans 9 pixels, the system isextremely stable. No realignment or refocusing is needed, althoughperiod checks of the alignment and focus, such as weekly checks, shouldbe performed.

[0133] A clear understanding of the noise sources for the array detectoris important, as the noise will ultimately determine the minimumbaseline fluctuation level and, thus, the LOD of the system. Dark noiseof the PDA can be attributed to dark current shot noise, diode resetnoise and circuit noise, which are not dependent on the number ofphotoelectrons generated in the diodes (i.e., the input lightintensity). According to the data given by the manufacturer, the darknoise (s_(d)) of the PDA is about 3,200 electrons at 0° C. Shot noise isgenerally defined as the combined noise associated with the randomgeneration of photons from the excitation source and the randomgeneration of photoelectrons in the diode junction, and is equal to thesquare root of the number of photoelectrons counted in each diode,(n_(e))^(1/2). The total rms noise level (s) of the PDA in the absenceof flicker noise (see below) can be expressed using the equation:

s=s _(d)+(n _(e))^(1/2).  (1)

[0134] Therefore, it is desirable to have as high a photon count aspossible. The electron well capacity of a diode is generallyproportional to the area of the sensing junction. A long but narrowdiode will maximize the dynamic range and the spatial resolution (in onedimension) at the same time. This comes with an increase in dark currentsuch that cooling becomes mandatory.

[0135] For the PDA used in this work, the saturation charge for eachdiode is about 25 pC, or 156 million electrons. This is almost threetimes as high as the PDA used in previous work (Culbertson et al., Anal.Chem. 70: 2629-2638 (1998)). In real absorption detection, however, thediodes should only be 85-90% saturated to allow room for baseline driftdue to uncontrollable variables over the period of data acquisition. Thetotal rms noise for an 85% saturated diode was calculated to be 14,700electrons according to Equation (1), given sd to be equal to 3,200electrons. Conversion of this value into absorbance units gave anabsorbance noise limit of 4.7×10⁻⁵.

[0136] Actual noise was measured from single-diode electropherograms.The tungsten lamp was used as the light source and was moved back andforth behind the capillary array to control the input light intensityand, thus, the number of photoelectrons generated at the diode junction.The measured rms noise level of one diode is linearly proportional tothe square root of the number of photoelectrons generated at the diodejunction. The intercept of the linear regression of the curve can berelated to the rms dark noise according to Equation (1). The measuredintercept value was 3,266 electrons, which was close to what was givenby the manufacturer, i.e., 3,200 electrons. Also, the measured slope,0.92, was close to the theoretical value of 1. When the diode was morethan 25% saturated with the tungsten lamp as the light source, the majornoise source was shot noise. This could be attributed to the relativelylow dark noise of the thermoelectrically cooled PDA and the superiorstability of the battery-powered DC tungsten lamp (thus a negligibleflicker noise). When a PDA was used at room temperature, the rms noiselevel was at least 5 times higher. The measured rms noise of the diodeat 85% saturation level can be converted to an absorbance unit of4.8×10⁻⁵, which is close to the expected noise limit of 4.7×10⁻⁵. Whenthe hand-held mercury lamp was used, the average measured rms noise forone diode was 9.0×10⁻⁵ at 85% saturation level, which was about twotimes higher than the expected value. This is believed to be due to theadditional intensity flicker noise associated with the mercury lamp.

[0137] Mathematical smoothing can reduce the noise significantly withoutdistorting the signal if properly used. To ensure that more data pointscan be used for smoothing, without sacrificing temporal response, ahigher data acquisition rate needs to be employed. For the PDA detector,data acquisition rate is limited by the digitization rate and theexposure time. The A/D converter in our system is capable of functioningat 25 kHz. So, 40 msec is the minimum exposure time for each data pointin the 1024 array. With the tungsten or mercury lamp as the light sourcein this experiment, a 40-msec exposure time was more than sufficient toattain around 85% saturation level for all diodes. Normally, an analytepeak is more than 10 sec in width, and 9 data points are enough torepresent a typical chromatographic or electrophoretic peak. So, up to25 data points (1 sec in time) can be used for smoothing with littlesacrifice of the width of the analyte peaks. Different kinds ofsmoothing approaches were compared, and boxcar smoothing proved to bethe most efficient method to suppress the noise here. After 25-pointboxcar smoothing, the average rms noise was lowered to 1.33×10⁻⁵ AU at85% saturation level with the tungsten lamp as the light source. One canconsider smoothing as increasing the dynamic range (electron well depth)of the diodes after the fact. The observed enhancement factor is closeto the factor of 5 predicted by Equation (1).

[0138] To probe the actual detection limits achievable using thiscapillary array absorption detection system, electrophoresis ofrhodamine 6G, at the concentration of 4×10⁻⁷ M in 1×TBE buffer solutionwas performed using 1×TBE as the running buffer. The sample washydrodynamically injected for 6 sec at a height difference of 8 cm. Nosample stacking was expected under these conditions. Capillaryelectrophoresis was run at 250 V/cm. Detection was performed at awavelength of 552 nm, defined by an interference filter with 10-nmbandwidth. The electropherogram from one capillary in the array is shownin FIG. 3, which is a graph of absorbance vs. frame number, in which thetop trace represents the raw data and the bottom trace represents thedata with a 25-point boxcar smoothing. The S/N for the rhodamine 6G peakwas about 8 (based on a peak height of 0.0002 and an rms noise betweenframe 1950 and frame 2250 of 2.6×10⁻⁵), which was near the detectionlimit predicted by Eq. (1). After 25-point boxcar smoothing, the S/Nratio was improved to about 45, as shown in FIG. 3. The resulting1.8×10⁻⁸ M LOD (S/N=2) for rhodamine 6G injected is comparable to whatmost commercial single-capillary machines could achieve.

[0139] The hand-held mercury lamp used in this experiment had much morefluctuation than the tungsten lamp did, but less than the pen-lightmercury lamp used in previous work (Culbertson et al. (1998), supra).This is inherent to the discharge nature of the mercury source ascompared to Joule heating in the tungsten source. While thebattery-operated tungsten lamp provided negligible flicker noise in thesystem, a double-beam scheme was employed to cancel the flicker noisedue to the mercury lamp. Certain capillaries in this 96-capillary arraywere injected with blank samples (buffer solution), and the signals fromthem were used as reference signals. Signals from other capillaries werenormalized to the level of the reference signal from the nearestreference capillary, and then the reference signal was subtracted fromthe normalized signals. FIG. 4, which is a graph of light intensity(counts) vs. frame number vs. value after subtraction (in counts), inwhich (A) is the electropherogram before noise cancellation, (B) is thereference signal from a blank capillary, and (C) is the electropherogramafter noise cancellation, shows the effect of the noise cancellationscheme for a signal at about 85% saturation level. The baseline driftand the intermediate-term noise (i.e., those on the time scale of thesignal peaks) were reduced. After normalization to the reference signal,the rms noise of the signal was lowered to 6.0×10⁻⁵ AU from 9.1×10⁻⁵ AU.The short-term (high frequency) noise was actually a bit higher.However, these were adequately smoothed out by the boxcar algorithmdescribed above. It was found that, in this 96-capillary array, theblank signal from one capillary could act well enough as the referencefor signals from ten capillaries on each side. So only five referencecapillaries were needed in the entire 96-capillary array. We note thatsince the data in each diode was acquired consecutively by thedigitizer, true temporal correlation of the flicker noise still does notexist between the reference and the measurement channels. Thiscontributes to the short-term noise. This aspect of the system couldpotentially be improved in the future with more sophisticated diodearrays with flexible clock functions.

[0140]FIG. 5, which is a graph of intensity vs. frame number, shows theresult of the MEKC separation of five neutral (polyaromatichydrocarbons) compounds, which are, in order, 9,10-diphenyl-anthracene(9×10⁻⁵M), benzo[ghi]perylene (1×10⁻⁴ M), benzo[a]pyrene (6×10⁻⁵M),benz[a]anthracene (4×10⁻⁵M), fluoranthene (1×10⁻⁴M) and anthracene(5×10⁻⁵M). The LOD (SIN=2) was 1.9×10-6 M before smoothing and 9.2×10-7M after smoothing. The final noise level was higher by about 2-foldcompared to the CZE separation experiment due to the higher intensityfluctuation of the hand-held mercury lamp, as discussed above.

[0141] The MEKC separation also generated very high current, which is 30μA per capillary and about 3 mA for the whole array. Therefore, a largeamount of heat was produced during the separation. Some coolingapproaches needed to be employed to help the heat dissipation. It wasfound that the hottest part in this setup during the separation was thedetection window. This was because all of the capillaries were denselypacked together in this region. To avoid mechanical vibrations in thecapillary array, which would bring about excess amounts of noise, alaminar flow of nitrogen gas was created in parallel to the detectionwindow of the capillary array to carry away the heat generated in thisregion. After the nitrogen cooling approach was employed, heatdissipation was not a problem in this setup.

[0142] To minimize the cross-talk between adjacent capillaries, theimage of a capillary on the face of the PDA needs to be big enough toensure that the diode corresponding to the center of the capillaryreceives minimal stray light from adjacent capillaries. It was found ifthe image of one capillary covers more than 8 diodes in the PDA,cross-talk was less than 0.2%, which is negligible for the multiplexedanalysis. Cross-talk was also found to be related to the spatialalignment of the capillary array. We found that the image of eachcapillary in the array needs to be parallel to the diodes in the PDA.Otherwise the signal from more than one capillary may cross over eachdiode (which is narrow but long) and cause more cross-talk. The arrayalso needs to be confined to a plane, or else imaging will not beuniform for each capillary. Finally, vibrations in the capillaries,especially when high voltage is applied or when cooling fans areimproperly situated, will cause additional flicker noise in the system.Rigid mounting of the array and of the optics is, therefore, critical.

[0143]FIG. 6, which is the result of CZE separation of four visible dyesin the 96 capillary array, in which the order of dyes is 5CF (4×10⁻⁵ M),6CF (4×10⁻⁵ M), F (8×10⁻⁵ M) and DADCF (1.2×10 ⁻⁴M), the horizontaldirection represents the location of the capillaries, the verticaldirection represents migration time from 5.3 to 7.0 min, the top plotrepresents intensity across the array, and the left plot representsintensity along one of the capillaries. Relatively uniform separationresolution and S/N distribution can be observed from the reconstructedimage file. Cross-talk between adjacent capillaries was not observable,as expected for this analyte concentration range.

[0144] The results for multiplexed CE indicate that the migration timesare highly nonuniform among the capillaries. This is to be expected fromthe absence of temperature regulation and variations in the columnsurfaces. We have demonstrated that an internal standardization schemecan be employed to normalize the results among the capillaries so thatthe migration times and the peak areas are reliable enough forhigh-throughput applications. FIG. 7, which is graph of migration timevs. capillary number in which the open symbols represent raw data andthe closed symbols represent data normalized by two internal standardsfor two fluoresceins, confirms that, by using the first and the lastpeaks as the two internal standards, both the migration times and thepeak areas become uniform across the array. For the second and thirdpeaks the relative standard deviations (RSDs) of migration times werereduced from 17% and 22% to 1.9% and 2.0%, respectively, and the RSDs ofpeak areas were reduced from 65% and 87% to 3.8% and 8.8%, respectively.In the case of peak areas, two capillaries (#23 and #45, see FIG. 8,which is a graph of peak area vs. capillary number in which the opensymbols represent raw data and the closed symbols represent datanormalized by two internal standards for two fluoresceins) dominated thecontributions to the RSDs. If these two capillaries were omitted fromthe statistical analysis, the RSDs were lowered to 2.9% and 5.3%,respectively. The fact that this normalization scheme (Xue et al., Anal.Chem. 71:2642-2649 (1999)) works equally well with the absorptiondetector here shows that the data in FIG. 7B are all within the linearrange of the detector. This is not surprising, since at no time did theintensity decrease by more than 15% (FIG. 6). For larger absorptions, alogarithmic correction will need to be applied to maintain a linearresponse.

[0145] This example demonstrates for the first time a high-throughputsystem for analyzing multiple samples simultaneously using absorptiondetection. Uniform separation efficiency and good S/N were obtained. TheLOD achievable for such a system, such as the 96-capillary arrayelectrophoresis system described above, is comparable to those forcommercial single-capillary electrophoresis machines using absorptiondetection. The separation of ionic and of neutral analytes weredemonstrated by zone electrophoresis and by MEKC, respectively.Consequently, the capillary array electrophoresis system can doeverything single-capillary electrophoresis absorption instruments cando, only with much higher throughput. Potentially, the present inventivesystem also can serve as an alternative to HPLC in many applications. Nomoving parts were used in this system. Once the positions of allcomponents are fixed, the only thing that needs to be adjusted is thefocal point of the camera lens, just like taking a picture, to get thebest focused image of the capillary array. One focused, no noticeablechanges in the system were observed over many days. Also, no lasers areused, thus the system should be smaller, more cost effective and easierto use and maintain than the multiplexed laser-induced fluorescence CEsystems. Besides, the analytes do not have to be fluorescent to bedetected. The absorption wavelength can be selected by simply changing afilter. Since the sample injection process involves only movingdifferent microtiter plates under the injection block and can be fullyautomated, it should be possible to obtain a true throughput that is 100times higher than what conventional single CE absorption determinationscan achieve.

Example 2

[0146] This example demonstrates the application of the presentinvention to genetic typing and diagnosis.

[0147] Based on the 96-capillary array electrophoresis system of Example1, DNA analysis protocols were designed to take advantage of capillaryarray gel electrophoresis and absorption detection based on the inherentspectral properties of the DNA bases and the fact that a 100-bp DNAcontains 100 absorbing units that can provide excellent net absorptivityfor sensitive detection. The method was tested on two broadly used PCRprotocols using typical concentrations of starting materials.

[0148] Samples were prepared as follows:

[0149] Polymerase Chain Reaction

[0150] 1. Multiplexed PCR for Variable Number of Tandem Repeats (VNTR)Loci

[0151] AmpliFLP D1S80 PCR amplification kit was purchased fromPerkin-Elmer (Foster City, Calif.). The kit included D1 S80 PCR ReactionMix (containing two D1S80 primers, AmpliTaq DNA polymerase and dNTPs inbuffer), MgCl₂ solution and Control DNA 3 (human genomic DNA of D1 S80type 18, 31 in buffer). The PCR mixtures used were as follows:

[0152] Positive Control: 20 μl of D1 S80 PCR Reaction Mix, 10 μl ofMgCl₂ solution and 20 μl of Control DNA3.

[0153] Negative Control: 20 μl of D1 S80 PCR Reaction Mix, 10 μl ofMgCl₂ solution and 20 μl of autoclaved DI H₂O.

[0154] The polymerase chain reactions were performed with the followingparameters: 30 cycles of denaturation at 95° C. for 15 sec, annealing at66° C. for 15 sec, and extension at 72° C. for 40 sec. The thermalcycler used was a Perkin-Elmer GeneAmp PCR system 2400.

[0155] 2. PCR for Human Immunodeficiency Virus (HIV)

[0156] The HIV testing kit (Perkin-Elmer) included positive control DNAthat includes all parts of the HIV-1 genome, negative control DNA, HIVprimers, AmpliTaq DNA polymerase, dNTPs, PCR reaction buffer and MgCl₂solution. The PCR mixtures used are listed in Table 1. The protocol forthe Perkin-Elmer GeneAmp thermal cycler is 40 cycles of denaturation at95° C. for 30 sec, annealing and extension at 62° C. for 1 min. Theannealing and extension temperatures were the same for thisamplification. TABLE 1 PCR mixtures for HIV amplification AdditionComponent Order Volume Final Concentration Autoclaved, deionized water 132.8 μl 10× PCR buffer II 2   5 μl 1× DNTPs 3   1 μl 200 μM each dNTPeach HIV-1 primer 1 (SK38) 4   1 μl 0.5 μM HIV-1 primer 2 (SK39) 5   1μl 0.5 μM AmpliTaq DNA polymerase 6  0.2 μl 1 unit 25 Mm MgCl₂ solution7   5 μl 2.5 mM Positive Control DNA or 8   1 μl 0.5 μg human NegativeControl DNA placental DNA

[0157] DNA Purification

[0158] All PCR products were purified with Microcon YM-30 centrifugalfilter devices (Millipore, Bedford, Mass.). After the purification,salts, dNTPs and most HIV-1 primers were eliminated from the DNAsamples.

[0159] The 96 capillary array electrophoresis system with photodiodearray absorption detection as described in Example 1 was used. ADC-powered mercury lamp (UVP Inc., Upland, Calif.) was used as the lightsource, which gave lower noise levels than the AC-powered mercury lampused in previous work. The absorption wavelength was set at 254 nm by aninterference filter (Oriel). The total length of the capillaries was 55cm, with 35-cm effective length. The capillary array was first flushedwith deionized water and then with 1 of 2% PVP at a pressure of 100 psi.While the injection ends were immersed in the buffer reservoir, 0.5 mlof 2% PEO (600,000 MW) sieving matrix was pushed into the capillarybundle at 100 psi. The procedure roughly took 20 min. After the gelfilling, ethidium bromide was added to the buffer reservoirs at theconcentration of 11 μg/ml. The system was then pre-run for 10 min withthe electric field strength at 150 V/cm. After the pre-run ethidiumbromide should have spread out evenly in the sieving matrix throughelectrical migration. Ethidium bromide is known to make the DNAfragments more rigid, thereby leading to sharper bands in CGE. It wasnot expected to alter significantly the absorption strength of the DNAfragments in this study. The samples were injected electrokinetically at150 V/cm for 15 sec. A field strength of 150 V/cm was employed for theseparation. The total current was about 620 μA during the separationprocess. Twelve different samples were used in the 96-capillary arrayelectrophoresis experiment, which are detailed in Table 2. Each type ofsample was injected into and run in eight different capillaries in the96-capillary array. TABLE 2 Sample description in the capillary arrayelectrophoresis Sample No. Description 1 4 μl of HIV-1 primer-1 (SK38) 24 μl of purified HIV-1 negative PCR product 3 4 μl of purified HIV-1positive PCR product 4 4 μl of 100 bp ladder 5 3 μl of purified HIV-1negative PCR product with 1 μl of 100-bp ladder 6 3 μl of purified HIV-1positive PCR product with 1 μl of 100-bp ladder 7 4 μl of purified D1S80negative PCR product 8 4 μl of purified D1S80 positive PCR product 9 4μl of 50-bp ladder 10 3 μl of purified D1S80 negative PCR product with 1μl of 50-bp ladder 11 3 μl of purified D1S80 positive PCR product with 1μl of 50-bp ladder 12 4 μl of DI H₂O

[0160]FIG. 9, which is a reconstructed two-dimensional electropherogramfor capillary array electrophoresis, in which the 12 capillaries(corresponding to the 12 samples described in Table 2) are alignedvertically and the migration direction is from left to right, shows theresult of the capillary array gel electrophoresis for DNA analysis as areconstructed “gel” image. The vertical direction represents thecapillary array arrangement, while the horizontal direction representsthe migration time. All separations were finished within 25 min.Capillary #84 (Sample type 11, see Table 2) showed bad separationresolution after 350 base pairs. The other 95 capillaries gavereasonable separation and good signal-to-noise ratios. The migrationtimes and peak intensities were highly non-uniform among thecapillaries. This was to be expected from the absence of temperatureregulation and variations in the column surfaces. An internalstandardization scheme can be employed to normalize the results amongthe capillaries so the migration times and the peak areas are reliableenough for high-throughput applications.

[0161] Actual electropherograms were extracted from capillaries #3, 9,17, 25, 33, 41, 49, 57, 65, 73, 85 and 89 to represent each type ofsample described in Table 2, and are shown in FIG. 10, in which the 12capillaries (corresponding to the 12 samples described in Table 2) arestacked vertically accordingly to the order in FIG. 9 and the migrationtimes are plotted from left to right. In capillaries #9-16 and #17-24,negative and positive HIV-1 PCR products were injected respectively. Thepositive and negative results can be easily differentiated through theHIV-1 gag fragment peak (triangle), which appeared only in theelectropherograms from capillaries #17-24. Both the positive andnegative HIV-1 PCR samples also contained the excess primers (circle)and the primer dimers (cross). To provide an even higher level ofconfidence for identification, despite the variation of migration timesamong capillaries, the HIV-1 gag fragments, primers and primer dimerscan be sized by mixing the PCR products with 100-bp DNA ladders(capillaries #25-32), and injecting them into capillaries #33-40 and#41-48. The electropherograms from the latter two groups of capillariesshowed that the HIV-1 gag fragment is about 115 bp and the primer dimeris about 60 bp.

[0162] In the electropherograms from capillaries #1-8, the HIV-1 primersgave broad peaks, which we believe are due to sample overloading.Deionized H₂O was injected into capillaries #89-96, which gave blankelectropherograms. These electropherograms served as blank referencesand were subtracted from the signals in the other capillaries to cancelout the flicker noise from the mercury lamp, as reported before. Theelectropherograms from capillaries #49-56 showed negative D1 S80 PCRresults, where only the primer peaks can be observed. Theelectropherograms from capillaries #57-64 showed positive D1 S80genotyping PCR results. Two component peaks (D1 S 80 type 18 and 31) canbe observed as expected from the heterozygous samples in addition to thevery broad primer peaks. Again, to increase the confidence level foridentification, the two D1 S80 components as well as the primer wereroughly sized by mixing each PCR product with a 50-bp ladder(capillaries #65-72) and injecting them into capillaries #73-80(negative) and #81-88 (positive). The results showed the two D1 S80components to be about 400-bp and 600-bp.

[0163] Current high-throughput approaches to the analysis of PCRproducts are based primarily on electrophoretic separation andlaser-excited fluorescence detection. This example demonstrates that thepresent invention can be applied to genetic typing and diagnosis basedsimply on UV absorption detection. The additive contribution of eachbase pair to the total absorption signal provides adequate detectionsensitivity for analyzing most PCR products. Not only is the use ofspecialized and potentially toxic fluorescent labels eliminated, butalso the complexity and cost of the instrumentation are greatly reduced.For example, no lasers are used. UV absorption detection of DNA productsreduces the cost of analysis since it does not require labeling. Thecapillary array was flushed with water in between runs and did not showany degradation over tens of runs in a one-month period. Since thesample injection can be fully automated, this example demonstrates thatit should be possible to obtain a true DNA analysis throughput that is100 times (scalable to 1,000 times) higher than what commercial singlecapillary gel electrophoresis systems can achieve, at relatively lowcost.

Example 3

[0164] This example demonstrates the application of the presentinvention to high-throughput comprehensive peptide mapping of proteins.

[0165] An experimental CE setup for multi-dimensional 96-capillary arrayelectrophoresis similar to that of Example 1 was used. Briefly, a totalof 96 fused-silica capillaries (Polymicro Technologies, Inc., Phoenix,Ariz.), 50-μm i.d. and 360-μm o.d., with 50-cm effective length and70-cm total length were packed side by side at the detection window andclamped between two flat surfaces of a plastic mount. The window wascreated after packing by using an excimer laser beam to burn off thepolyimide coating. At the ground end (outlet), every 12 capillaries werebundled together to allow simultaneous filling of six-different buffersfor six-dimensional peptide mapping. At the injection end, the capillaryarray was spread out and mounted on a copper plate to form an 8×12format with dimensions to fit into a 96-well microtiter plate for sampleintroduction. Gold-coated pins (96) (MillMax Mfg. Corp.) were mounted onthe copper plate near the capillary tips to serve as individualelectrodes, with the capillary tips slightly extended (˜0.5 mm) beyondthe electrodes to guarantee contact with small-volume samples. Ahigh-voltage power supply (Glassman High Voltage, Inc., WhitehorseStation, N.J.) was used to drive the electrophoresis.

[0166] The light source, filter, capillary array holder, and PDAdetector were all contained in a light-tight metal box attached to anoptical table as described above. As the light source, a 213.9-nm zinclamp (model ZN-2138, Cole-Parmer) was used for UV absorption detection.The transmitted light from the capillary array passed through aninterference filter (Oriel) and a quartz lens (Nikon; f.l.=105 mm;F=4.5). An inverted-image of the capillary array at a nominalmagnification factor of 1.2 was created by the quartz lens on the faceof the PDA. The PDA (Hamamatsu model S5964, Hamamatsu, Japan)incorporated a linear image sensor chip (1024 diodes, 25-μm in width,2500-μm in height), a driver/amplifier circuit, and a temperaturecontroller. The built-in driver/amplifier circuit was interfaced to anIBM-compatible computer (233-MHz Pentium, Packard Bell) via a NationalInstrument PCI E Series multifunction 16-bit I/O board. All codes usedto operate the PDA and to acquire the data were written in house usingLabview 5.0 software (National Instruments, Austin, Tex.).

[0167] The raw data sets were converted into single-diodeelectropherograms by another in-house Labview program. Data treatmentand analysis were performed using Microsoft Excel 97 and GRAMS/32 5.05(Galactic Industries).

[0168] In the multi-dimensional CE experiments, the capillary array wasfirst flushed with methanol and then water for cleanup. The six runningbuffers used for four-dimensional CZE separations and two-dimensionalMEKC separations were as follows: (1) 50 mM Trizam®·Phosphate buffer (pH2.5 with H₃PO₄), (2) 50 mM sodium acetate buffer (pH 5.0 with aceticacid), (3) 0.1 M Trizma®·Base/0.1 M Tricine buffer (pH 8.1), (4) 0.1 MCHES/0.1 M NaOH (pH 9.3), (5) 0.1% Tween 20 in 50 mM sodium acetatebuffer (pH 5.0 with acetic acid), and (6) 7% Tween 20 and 10 mM SDS in0.1 M Trizma®·Base/0.1 M Tricine buffer (pH 8.1). The samples were putinto a 96-well microtiter sample plate (1 μl/well) and gravity injectedat the anode for 60 sec at 8-cm height. The applied electric field was+157 V/cm and electrophoresis was performed at ambient temperature.After each run, the capillaries were rinsed with 0.1 M NaOH, water, andrunning buffer for 5 min each.

[0169] All CE separations for CZE and MEKC analyses were optimized on anISCO (Lincoln, Nebr.) Model 3140 Electropherograph System before themulti-dimensional multiplexed CE runs. Bare fused-silica capillaries(Polymicro Technologies, Inc., Phoenix, Ariz.) with 50-cm effectivelength and 75-cm total length (50-μm i.d. and 361-μm o.d.) were used.Four different buffer systems were investigated for CZE separations andtwo different buffer systems were also investigated for MEKCseparations. The optimal compositions are described above. The sampleswere introduced with hydrodynamic flow by placing the inlet of thecapillary into the sample vial and raising the sample vial 30 cm abovethe exit vial and allowing the sample to siphon into the capillary for10 sec. The applied electric field was +227 V/cm and electrophoresis wasperformed at ambient temperature. The detection wavelength was set at214 nm for monitoring peptide fragments. After each run, the capillarywas rinsed with 0.1 M NaOH, water, and running buffer in order for 5 mineach.

[0170] Tryptic digestion of BLGA and BLGB was carried out according tothe procedure of Cobb et al. ((1989), supra) without the dialysis andlyophilization steps. A mixture of 2 mg/ml BLG and trypsin was preparedwith a 10 mM Trizma®·Base and 50 mM ammonium acetate buffer (pH 8.2)containing 0.1 mM calcium chloride. Trypsin was added at atrypsin-protein ratio of 1:50 (w/w), and the digestion mixture wasincubated at 37° C. for 5 hr. The digest was directly injected into theseparation CE system without filtration.

[0171] Bovine β-lactoglobulin (BLG) is the major whey protein of cow'smilk. Mature bovine BLG has 162 residues as shown in FIG. 11 [SEQ ID NO:1], which represents the peptide maps of three variants of BLG. Threevariants of BLG, labeled as A, B, and C, commonly occur in cow's milk.Variants A and B differ at two sites: aspartic acid (D) 64 in BLGA ischanged to glycine (G) in BLGB, and valine (V) 118 in BLGA is changed toalanine (A) in BLGB. Variants B and C differ at one site: glutamine (O)59 in BLGB is changed to histidine (H) in BLGC (Bin et al., ProteinScience 8: 75-83 (1999)). Tryptic digestion is quantitative and veryspecific because trypsin cleaves only at the C-terminal side of lysineand arginine residues. The theoretical fragments are listed in Table 3.In the case of BLG, seventeen different peptides exist after trypticdigestion. TABLE 3 Theoretical products of tryptic digest of BLGA andBLGB Expected fragment Sequence Residues 1 L-I-V-T-Q-T-M-K 1-8 2G-L-D-I-Q-K  9-14 3 V-A-G-T-W-Y-S-L-A-M-A-A-S-D-I-S-L-L-D-A-Q-S-P-L-R15-40 4 V-Y-V-E-E-L-K-P-T-P-E-G-D-L-E-T-L-L-Q-K 41-60 5W-E-N-D-E-C-A-Q-K (W-E-N-G-E-C-A-Q-K)^(α) 61-69 6 K 70 7 I-A-A-E-K 71-758 T-K 76-77 9 I-P-A-V-F-K 78-83 10 I-D-A-L-N-E-N-K 84-91 11V-L-V-L-D-T-D-Y-K  92-100 12 K 101 13Y-L-L-F-V-M-E-N-S-A-E-P-E-Q-S-L-V-C-Q-C-L-V-R 102-124(Y-L-L-F-V-M-E-N-S-A-E-P-E-Q-S-L-A-C-Q-C-L-V-R)^(α) 14T-P-E-V-D-D-E-A-L-E-K 125-135 15 F-D-K 136-138 16 A-L-K 139-141 17A-L-P-M-H-I-R 142-148 18 L-S-F-N-P-T-Q-L-E-E-Q-C-M-I 149-162

[0172] A multiplexed capillary array system allowed high-throughputcharacterization and generation of peptide maps of proteins, afterenzymatic digestion, using six-dimensional capillary electrophoresis ata constant applied electric field. In a 96-capillary array imageobtained using a zinc lamp and the PDA, the center of each capillarycorresponds to a “peak” in the image. Between every two center “peaks,”there is a “valley” which corresponds to the wall of the capillary. Whenthe capillary array image was well-focused onto the PDA, the intensitiesof these valleys became minimized. This feature was used to produce thebest focusing. “Spacing peaks” (see above) were eliminated in thisstudy. This results from a special treatment on the window of thecapillary array, whereby epoxy glue was applied between the capillarieson the detection window. The epoxy glue greatly strengthened the windowarea of the capillary array and minimized movement of the capillaries inthe electric field. Because the epoxy glue is not UV transparent, itabsorbed all of the light that would have passed through the spacing ofthe capillaries and eliminated the “spacing peaks.” This further reducedstray light for absorption detection. The zinc lamp provided 213.9-nmlight that is well-suited for the absorption detection of peptides. Theemitting length of the zinc lamp is about 2 cm, which is long enough foruniform illumination of the entire capillary array (1.5 cm). There wasless than 2× variation in optical throughput from the center to the edgeof the array. This means that the detection limit varied by less than{square root}{square root over (2)}× across the array. The zinc lamp wasvery stable and produced negligible flicker noise, so no double-beamsubtraction was necessary in this experiment.

[0173] Up to the present, peptide mapping of proteins is primarily basedon the cleavage of proteins with enzyme or chemical agents, followed bya one- or two-dimensional separation of the resulting peptide fragments.While the resolution of peptide fragments achieved by one-dimensionalseparation is often insufficient to resolve the complex mixture ofpeptides, the conventional two-dimensional techniques suffer from thedifficulty of efficiently recovering uncontaminated peptides from thefirst dimension to transfer to the second dimension. Also,two-dimensional separation conditions have to be changed according tothe sample protein. To overcome these problems a six-dimensional systemwas used. By combining four different CZE conditions at different pHsand two different MEKC conditions, comprehensive and complementaryinformation about the peptides of arbitrary proteins was obtained. Asshown in the results of single-capillary runs (FIGS. 15 and 16), thepeptide fragments at different separation conditions showed differentbut related peptide maps. FIG. 12 shows the results of thesix-dimensional separations of tryptic digests of BLGA and BLGB in the96-capillary array, in which (A) is 50 mM Trizam®•phosphate buffer (pH2.5 with H₃PO₄), (B) is 50 mM sodium acetate buffer (pH 5.0 with aceticacid), (C) is 0.1% Tween 20 in 50 mM sodium acetate buffer (pH 5.0 withacetic acid), (D) is 0.1 M Trizma®•Base/0.1 M tricine buffer (pH 8.1),(E) is 7% Tween 20 and 10 mM SDS in 0.1 M Trizma®•Base/0.1 M tricinebuffer (pH 8.1), and (F) is 0.1 M CHES/0.1 M NaOH (pH 9.3). The verticaldirection represents the capillary array arrangement, while thehorizontal direction represents the migration time. The applied electricfield was +157 V/cm. A column, bare fused-silica capillary witheffective/total length of 50/70 cm and 50 μm i.d. was used. Hydrodynamicinjection was conducted for 60 sec at 8 cm height.

[0174] In the single-capillary CE system, when the electric field of+227 V/cm was applied to the 50-μm i.d. capillary, the current was below20 μA at ambient temperature. However, the capillaries were packed sideby side in the 96-capillary array system and the dense packing generateda much higher temperature at the same separation condition.Consequently, some of the capillaries can lose current during theseparation because of the formation of bubbles. Thus, a lower electricfield of +157 V/cm was applied in spite of an increased analysis time.All separations were still completed within 45 min. Although capillaries87 and 88 showed unusually long separation times and much lower signallevels, all of the other 94 capillaries gave comparable separation timesand good signal-to-noise ratios. As shown in the reconstructed image,even for the same separation condition, the migration times and peakintensities were not uniform among the capillaries. This phenomenon isto be expected from the absence of temperature control in the experiment(Xue et al. (1999), supra; and Gong et al. (1999), supra). However, wehave demonstrated that an internal standardization scheme can be appliedto normalize the results among the capillaries (Xue et al. (1999),supra; and Gong et al. (1999), supra) so that the corrected migrationtimes and peak areas are of sufficient reliability for high-throughputapplications.

[0175]FIGS. 13 and 14 show the extracted electropherograms of BLGA andBLGB, respectively, as derived from the six-dimensional data in FIG. 12.The individual maps are virtually identical to the single-capillaryresults. The peptide patterns of BLGA and BLGB can be easilydifferentiated in each of the six-dimensional separation conditions.Compared with the single-capillary run, 0.1% Tween 20 in 50 mM sodiumacetate buffer (pH 5.0) gave slower migration times in the 96-capillaryarray. In part, this was due to the lower applied electric field (+157vs. +227 V/cm). Tween 20 (0.1%), instead of Tween 20 (0.2%), at the MEKCcondition was used in the array because the latter would have resultedin even longer analysis times.

[0176] Since peptides are polymers of amino acids, they typically have alimited number of charged states in their structure depending on thepresence of amino acid moieties with ionizable side chains (Landers etal., Handbook of Capillary Electrophoresis, CRC Press (1997), pp.219-221). This determines the pH ranges for CE separations beyond whichno theoretical optimization can be performed. At pH<2, all ionizablegroups of peptides will be protonated. The number of basic residues inthe peptide chain will determine the overall charge-state of themolecule. At pH>10, all ionizable groups will be de-protonated,resulting in a negatively charged peptide. At these extreme pHconditions, the separation of peptides cannot be adjusted. Atintermediate pHs, partly ionized termini and side chain residues allowoptimization of the peptide separation. Therefore, four different pHconditions, i.e., pH 2.5, 5.0, 8.1, and 9.3, were selected for theseparation of peptide fragments. Since ionization of peptides generallyoccurs over a pH range of 2.5-3.0, these pHs are sufficiently far apartto give independent electropherograms but are close enough to form acontinuous (combinatorial) set of conditions.

[0177]FIG. 15 shows typical peptide maps of BLGA and BLGB at four pHconditions for CZE using a single capillary after tryptic digestion, inwhich (A) is 0.1 M CHES/0.1 M NaOH (pH 9.3), (B) is 0.1 MTrizma®•Base/0.1 M tricine buffer (pH 8.1), (C) is 50 mM sodium acetatebuffer (pH 5.0 with acetic acid), and (D) is 50 mM Trizma®•phosphaebuffer (pH 2.5 with H₃PO₄). The applied electric field was +227 V/cm.Separation was at ambient temperature. A column, bare fused silicacapillary with effective/total length of 50/75 cm and 50 μm i.d. wasused. Hydrodynamic injection was conducted for 10 sec at 30 cm height.All peptide peaks at the four different separation conditions werewell-resolved within about 30 min. Each condition revealed differentpeptide maps. The separation of peptides above pH 5.0 were completedquickly and efficiently within 15 min (FIG. 15A, B and C). Theelectropherograms at pH 2.5 showed good resolution in spite of arelatively long analysis time (FIG. 15D). Adsorption of the proteins onthe capillary wall in CE can be a serious problem. This can lead tovariable migration times, band broadening, and peak tailing. At pH 2.5,much of the negative charge had been titrated off the silica walls ofthe capillary such that there was little coulombic interaction betweenthe peptide and the wall. This is not true of other pH conditions.Therefore, high ionic strengths were used in the running buffers toprovide efficient separations because of reduced interaction between thepeptide fragments and the capillary wall.

[0178]FIG. 15 (FIG. 15A at pH 9.3; FIG. 15B at pH 8.1; FIG. 15C at pH5.0; and FIG. 15D at pH 2.5) shows the differences in the peptide mapsof BLGA and BLGB at four different CZE conditions. Although the aminoacid sequences of bovine BLGA and BLGB differ only at two sites (64 and118, FIG. 11, SEQ ID NO: 11) among the 162 amino acids, the differencesin the peptide fragments of BLGA and BLGB could be clearly identified.Peptide mapping by CE is increasingly being utilized as a complement, ifnot a viable substitute, for the already established technique of HPLC.CE has several advantages over HPLC, including much higher efficiencyand a smaller sample requirement. Moreover, CE also offers astraightforward correlation of migration time with physiochemicalproperties. According to the dependence on pH, the net charges of eachpeptide fragment can be confirmed. Interpretation of the combinedpeptide maps at the four different conditions (FIGS. 15A-D) showedcomprehensive data with overlapping redundancy for the peptides thatcannot be obtained using only one separation condition.

[0179] The addition of surfactants to the running buffer can add severalnew aspects to the separation mechanism. Above their critical micelleconcentration, the surfactants form micelles, introducing apseudo-stationary phase into the running buffer. FIG. 16 shows MEKCpeptide maps of BLGA and BLGB obtained at two different MEKC conditionsusing a nonionic surfactant, Tween 20 (i.e., 0.2% Tween 20 in 50 mMsodium acetate buffer (pH 5.0 with acetic acid) (A and B), and/or thecombination of nonionic and anionic surfactant, Tween 20+SDS (i.e., 7%Tween and 10 mM SDS in 0.1 M Trizma®•Base/0.1 M tricine buffer (pH 8.1)(C and D). The use of the surfactants in the intermediate-pH range wasappropriate because more neutral peptides should exist there than at theextreme pHs such as pH 2 or 10.

[0180] In the MEKC conditions using a nonionic surfactant (MEKC Iconditions) (FIG. 16, A and B), the electropherograms showed higherresolution compared to the electropherograms obtained using CZEconditions (FIG. 15C). There are some minor shifts in relative peakpositions, mostly for the early eluting components. At pH 5.0, as theconcentration of the nonionic surfactant Tween 20 is increased, theresolution of peptide fragments increased without an increase in thecurrent. Although all of the peptide fragments showed baselineseparation above 0.3% Tween 20, the analysis time was approximately 1hr. Under the applied electric field of +227 V/cm, 0.2% Tween 20 wassufficient to improve the resolution compared to CZE. This buffer wasselected so that the analysis time can be kept under 20 min.

[0181] In the MEKC conditions using the combination of nonionic andanionic surfactants (MEKC II conditions) (FIG. 16, C and D), the higherconcentration of Tween 20 and the anionic surfactant SDS were needed toincrease the resolution. Because higher pH caused larger mobilities forthe peptide fragments in solution, a lower frequency of dynamicpartition into the micelles resulted. However, as the concentration ofsurfactants in the running buffer is increased to favor partition, theanalysis time also increased and the sensitivity decreased. So, 7% Tween20 and 10 mM SDS were chosen as optimal surfactant concentrations forMEKC II. The unique advantage of combining Tween 20 and SDS led to thefull resolution of all peptide fragments, enabling higher resolution forboth neutral peptides and same-charged peptides. FIG. 17 shows theeffect of Tween 20 concentration at pH 8.1 on the MEKC peptide maps ofBLGB in terms of migration time (min). It is clear that both resolutionand selectivity are affected by the surfactant. The patterns of thepeptide maps obtained at the six different CE conditions above (FIGS. 15and 16) were reproducible as judged by comparing the results of fiverepeated injections in each case.

[0182] This example demonstrates high-throughput, multi-dimensionalpeptide mapping of proteins in accordance with the present invention,such as by using multiplexed capillary electrophoresis. Uniquefingerprints of closely related sample proteins, BLGA and BLGB, on a96-capillary array with six different separation conditions wereobtained within 45 min. The 214-nm monitoring of peptide bonds isuniversal but also very complex because a typical map generally contains20-150 peaks (Dong et al. (1992), supra) and all of the fragments shouldideally be totally resolved. Maps of unknown proteins are notunambiguous by using only one- or two-dimensional separation. With sixcomplementary separation conditions, there is no need to reoptimize theprotocol for each new sample. The instrumental set-up is simplified andautomation of the method becomes possible. Most importantly, thismulti-dimensional and multiplexed peptide mapping technique isinherently a small-volume and high-throughput approach. For example,although only two proteins are studied here, sixteen different proteinscan be mapped at the same time starting with μl samples. Alternatively,different enzymes or chemical agents can be employed to providecomplementary sets of peptide maps for further confirmation. For complexunknown samples, internal standards can be used to normalize themigration times and peak areas (Xue et al. (1999), supra; and Gong etal. (1999), supra). The six separation conditions then can be used torank order the peptide fragments with respect to their isoelectricpoints (pIs), very much like a high-resolution gradient elution. The useof different sets of buffer systems in each capillary in the array is ineffect a combinatorial approach to developing the best separationconditions for a given group of analytes. This last feature should begenerally useful in all applications of CE. Finally, capillary arraysare compatible with on-column digestion of proteins (Chang et al., Anal.Chem. 65: 294-2951 (1993)) so that full automation in multiple channels(Chang et al. (1992), supra; Zhang et al. (1999), supra; and Zhang etal., Anal. Chem. 71: 1138-1145 (1999)) is possible with sub-microlitervolumes of samples and reagents.

Example 4

[0183] This example demonstrates the use of the present invention incombinatorial screening of enzyme activity.

[0184] The multiplexed capillary system described above was used. Atotal of 96 fused-silica capillaries (50-μm i.d., 150-μm o.d.; PolymicroTechnologies, Phoenix, Ariz.) packed side by side with 50-cm effectivelength and 70-cm total length were used for separating reactants,products and enzymes. Preloaded (0.2 ml) 96-well plates were used asreactors for carrying out the enzyme reactions. A 254-nm mercury lampwas used for UV absorption detection. A voltage of +11 kV (˜157 V/cm)was applied across the capillaries for separation.

[0185] During the period of incubation, the plates were covered byplastic film to minimize evaporation of the reaction solution. Thisallowed the concentrations of enzyme and substrate to remain as stableas possible.

[0186] Because of the inhibition of pyruvate on the catalysis of LDH, anoptimal concentration of pyruvate is important. At pH 7.2 and 25° C., 2mM pyruvate is normally suitable for the M₄ isoenzyme. So, in thereaction buffers (20 mM phosphate, pH ranges from 5.8˜8.0), 2.0 mM NADHand ˜2.0 mM pyruvate were added as the substrates for the enzymaticreaction. The direction of reaction was chosen as follows:${NADH} + {{pyruvate}\overset{LDH}{}{lactate}} + {{NAD}^{+}.}$

[0187] The reaction was allowed to proceed for a fixed period prior tohydrodynamic injection of the reactants, products and enzymes into thecapillaries. A solution of 10 mM phosphate with pH of 8.0 was used asthe separation buffer. After applying voltage, all components werereadily separated due to different mobilities. Since low concentrationsof enzyme (5×10⁻¹⁰˜1×10⁻⁸ M) (pseudo-first-order reaction) were used,the amount of NAD⁺ formed during a given period of time at a fixedtemperature is linearly proportional to the LDH activity. Therefore, theLDH activity can be quantified by measuring the peak area of NAD⁺ formedduring the fixed incubation period. The areas of the NAD⁺ peak and theNADH peak were integrated. Because NAD⁺ and NADH both absorb at 254 nm,while the enzyme does not contribute much to the background at thiswavelength, a 254-nm mercury lamp was used. By measuring the absorptioncoefficients of NAD⁺ and NADH at 254 nm in a conventionalspectrophotometer, the peak areas were converted to amounts. The ratiobetween the NAD⁺ amounts formed and the original NADH amounts wascalculated. Since a small background reaction exists, blank reactionswere monitored and subtracted.

[0188] Separate 20 mM phosphate buffers with pH values of 5.8, 6.3, 6.5,6.7, 7.0, 7.3, 7.6 and 8.0 were prepared. Buffer solution (175 μl), 10μl enzyme solution, 10 μl 40 mM NADH and 5 μl 90 mM pyruvate were addedinto 96 wells for reaction. Reactions progressed at room temperature.The various final concentrations of enzyme in those solutions were5×10⁻¹⁰ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M,8×10⁹ M and 1×10-8 M. Thus, for every concentration of enzyme, therewere 8 different buffer solutions. At the same time, for every buffersolution with a different pH value, there were 9 differentconcentrations of the enzyme. The reaction mixtures were incubated for30 min, 78 min, 128 min, 180 min, 308 min, 420 min, 480 min and 1,477min. After each incubation period, hydrodynamic injection was used toinitiate CE analysis. The volumes withdrawn each time from the reactionvials were negligible compared to the starting volumes. Therefore,essentially non-intrusive monitoring was achieved.

[0189] The reason for using hydrodynamic injection is thatelectrokinetic injection would have caused serious errors. This isbecause of different surface conditions from capillary to capillary,different compositions of the buffer solutions, different capillarytemperatures, and different migration velocities of NADH and NAD⁺ (Leeet al., Anal. Chem. 64: 1226-1231 (1992)). The effect is obvious from acomparision of FIG. 18A, which is a graph of the ratio of the amount ofNADH (injected) to the amount of NAD (injected) vs. the results fromnine electrokinetic injections, and FIG. 18B, which is a graph of theratio of the amount of NADH (injected) to the amount of NAD (injected)vs. the results from nine hydrodynamic injections. There, a solutionwith 1 mM NADH and 1 mM NAD⁺ was prepared and injected separately byhydrodynamic injection and electrokinetic injection into 9 differentcapillaries. A 5 cm difference in height was maintained for 60 sec forthe former and +11 kV was applied for 30 sec for the latter. By applying+11 kV to the capillaries, the NADH and NAD⁺ sample zones were separatedand driven across the detection windows. Since all of the solutions hadthe same NADH and NAD⁺ concentrations, identical peak areas wereexpected for the NADH peaks and NAD⁺peaks. According to FIG. 18,hydrodynamic injection only caused a very small standard deviation (SD),which was about 0.027 with a mean of 0.817, while electrokineticinjection caused serious errors, with an SD of 0.483 and a mean of 3.66.Such an injection problem is more serious in capillary arrays comparedto repeated injections in a single capillary because of surfaceheterogeneity.

[0190] The migration times varied from capillary to capillary, becausethe conditions of the capillaries were different. Despite the differentmigration times, the NADH and NAD⁺ peaks were easily identified in thissimple mixture. These peak areas were used for quantitation. After eachincubation period, the fraction of NADH converted to NAD⁺ was calculatedusing the following equations:${{amount}\quad {of}\quad {NADH}\quad ({reacted})} = {{NAD}^{+}\quad {area} \times \frac{ɛ_{NADH}}{ɛ_{{NAD}^{+}}}}$${{fraction}\quad {of}\quad {NADH}\quad ({reacted})} = {\frac{{amount}\quad {of}\quad {NADH}\quad ({reacted})}{{{amount}\quad {of}\quad {NADH}\quad ({reacted})} + {{NADH}\quad {area}}}.}$

[0191] As stated before, the fraction of NADH (reacted) then could beused to represent the activity of the enzyme whenever this reaction ispseudo-first-order. The use of a ratio avoids problems with variationsin detection sensitivity and injected amounts among capillaries.Additional corrections for the different speeds of the analytes passingthe detector were made since hydrodynamic injection was employed (Lee etal. (1992), supra).

[0192] Since 8 pH conditions and 9 enzyme concentrations were screened,there were a total of 72 channels (capillaries) where products weredetected. In addition 16 channels of background reaction (8 pHconditions with substrates but no enzymes) were monitored. The other 8channels in the array were filled with the buffer (no substrate and noenzyme) as the absorption reference. The entire data set for the96-capillary array is shown as a reconstructed image in FIG. 19, whichis a reconstructed absorption image of combinatorial screening of enzymeactivity in a 96 capillary array in which the capillaries (1-96) arearranged from top to bottom and migration time (0-33 min) is plottedfrom left to right. The change in electroosmotic flow from one capillaryto the next and temperature variations are the main reasons forsubstantial variations in migration times among the channels. Evenlarger variations are expected because the sample pH and sample ionicstrengths are all different. The capillary walls will become dynamicallyaltered as a result of injection. Internal standards can be employed tonormalize the migration times, but were not needed for the simpleelectropherograms in this study. The intensity variations in FIG. 19 arepartly due to uneven illumination but mostly due to the expectedvariations in the extents of reaction in each channel.

[0193] Extracted electropherograms for activity screening are shown inFIGS. 20 and 21. At a constant pH (FIG. 20), it is easy to see that theextent of reaction increases with LDH concentration (left peak is NAD⁺(product); right peak is NADH (reactant); from top to bottom, theconcentrations are 0, 0.5 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nMand 10 nM; the enzyme is not detected at this concentration). At a fixedLDH concentration, i.e., 5×10⁻⁹ M (FIG. 21), it can be seen that thereis an optimum pH where the LDH activity is the highest (left peak isNAD⁺ (product); right peak is NADH (reactant); from top to bottom, thepH are 5.8, 6.3, 6.5, 6.7, 7.0, 7.3, 7.6 and 8.0; the enzyme is notdetected at this concentration).

[0194] The quantitative optimization results shown in FIG. 22, which isa graph of NADH conversion percentage vs. pH for series 1-9 at 180 minincubation, need to be examined more carefully by repeated sampling ofthe reaction mixture at well-defined time intervals. For short reactiontimes (FIG. 23, which is a graph of reaction percentage vs. pH forseries 1-9 for 30 min of LDH catalysis), there is a linear increase inthe extent of reaction as a function of LDH concentration for all pHconditions. This is ideal behavior. For long reaction times (FIG. 24,which is a graph of reaction percentage vs. pH value for series 1-9 for24 hr of LDH catalysis), nonlinearity is observed, especially when thefraction reacted exceeds 0.6. The reason for this is saturation of thereaction. When a significant fraction of the reagent (NADH) is consumed,the pseudo-first-order reaction description fails. The remedy is to usehigher reagent concentrations or to stay with short reaction times. Thisfeature shows the importance of monitoring the full kinetics ofreactions as opposed to single-point monitoring. FIG. 24 by itself wouldhave led to the incorrect conclusion that there is not much differencein enzymatic activity over a broad pH range.

Example 5

[0195] This example demonstrates the use of the present invention incombinatorial screening of homogeneous catalysis and the optimization ofa homogeneously catalyzed synthetic organic reaction.

[0196] The present inventive method was used to analyze a newpalladium-catalyzed annulation reaction (Zhang et al., J. Organometal.Chem. 576: 111-124 (1999)), which readily affords γ-carbolines,noteworthy for their biological activity. The optimal reactionconditions and the regiochemistry for this type of annulation aregenerally highly dependent on the nature of the palladium catalyst andthe base employed. Previous efforts to optimize this process employed 5%Pd(OAc)₂, 10% PPh₃ and Na₂CO₃ as base and afforded a 1:1 ratio ofisomers A/B in essentially a quantitative yield.

[0197] The nature of this and other catalytic reactions is that a lot ofparameters can affect the yield and “optimum” conditions are often foundby trial and error. The above reaction was run using 0.25 mmole in 5 mlof dimethyl formamide (DMF). The volume was reduced to 120 μl by using 6mm O.D. glass tubes sealed at one end and arranged in a 96-well format.The individual components were added as a DMF solution or as a slurry bypipetting. Septums were used to cap the reaction tubes to preventevaporation. All reactions were thus run on a 5 μmole scale. Heating wasprovided by a dry heat bath kept at 110° C. As an internal standard, 1μmole of norharman was added to the reaction mixture. No catalyticeffect on the system from the addition of the norharman was observed incontrol experiments. FIG. 25 shows the separation of the two isomericforms (A and B) of the product from the reagents and the internalstandard using two different buffers (40 mM NH₄OAc and 0.75% formic acidin methanol for 1a; 40 mM NH40ac and 0.75% formic acid in 80% DMf/20%H₂O for 1b), with an applied electric field of 140 V/cm, using bare,fused-silica capillaries with an effective/total length of 50/75 cm and50 μm I.D. hydrodynamic injection for 15 sec at 8 cm height. Ethanol andpure DMF were also tested, but the separation was not acceptable. Nobubbles were found in CAE, even when a low boiling point solvent, suchas methanol, was used.

[0198] One important feature of the experimental protocol is that thereaction mixture was injected into CAE without diluting or quenchingbefore analysis. At predetermined times during the reaction, thereaction block was removed from the heating platform, quickly cooled andput under the injection ends of the capillary array. No deleteriouseffect on the catalytic system was observed by this operation. Byavoiding sample manipulation (e.g. by pipetting out of the reactionvials), errors associated with transfer and contamination can bereduced. The CAE running buffer should be compatible with the reactionbuffer for hydrodynamic injection. When using methanol as the buffer,injection was not uniform. Only about half of the 96 capillaries hadadequate signal. It was not possible to increase the injection time,because some capillaries then became overloaded. When DMF-based bufferwas used, all 96 channels had uniform signal over three consecutiveruns. This buffer compatibility issue for CAE may be attributed to thedifferences in solution properties, such as viscosity and surfacetension, and was not observed in single-capillary experiments. The totalanalysis time is typically 60 min, plus 30 min for capillary cleaning.Judging from the resolution in FIG. 25, the capillaries could have beenshortened to 25% of the effective length to provide analysis times of 15min.

[0199] By choosing 8 different Pd catalysts and 11 different bases, 88different combinations were tested. FIG. 26 shows such a 96-capillaryseparation for the reaction conditions for 1 b in FIG. 25 and ahydrodynamic injection of 1 min, in which the horizontal direction spans88 capillaries (the remaining 8 capillaries contained solvent only andwere not plotted) and the vertical direction represents time.Information on the total yield (FIG. 27, which is a 3-dimensional bargraph of yield vs. catalyst vs. base, for reaction after 17 hr at 110°C., in which dppe is bis(diphenylphosphino)ethane, TABC istetra-n-butylammonium chloride, DABCO is 1,4-diazabicyclo[2.2.2]octane,and dba is trans, transdibenzylidene-acetone), selectivity (FIG. 28,which is the selectivity plot of two isomers produced by the reactions,wherein P1/P2 is the ratio of the two isomers A and B, respectively) andreaction kinetics (FIG. 29, which is a line graph of fractionalconversion vs. time (hr) vs. base, for the reaction using Pd(PPh₃)₄ asthe catalyst and various bases) can be obtained from theelectropherograms. By using Pd(OAc)₂ with the ligand PPh₃ as catalystand Na₂CO₃ as the base, a total yield of 84% was achieved with virtuallyno regioselectivity in the microreactor, compared with a quantitativeconversion (90% after 17 hours) with no selectivity under the protectionof N₂ in a 5 ml reaction. Among all of the bases, inorganic bases provedto be more effective in promoting the reaction. When pyridine or otherorganic bases were used, the yield was low and some side productsappeared. The ability to detect side products is clearly an advantage ofCAE. Preliminary results also reveal several new conditions which arequite effective in this annulation reaction. They are Pd(PPh₃)₄ withNa₂CO₃ (C9, 74%), Pd(dba)₂ with K₂CO₃ (E10, 72%), PdBr₂ plus 2PPh₃ withNa₂CO₃ (G9, 88%) and PdBr₂ plus 2PPh₃ with K₂CO₃ (G10, 96%). The lattertwo are in fact superior to the previous best catalytic condition (Zhanget al. (1999), supra). Complete regioselectivity is not observed in anyof the test conditions (FIG. 28), even though some prove to be betterthan other systems. The conditions G2, H2, and B1 have some selectivity,but unfortunately their yields are low. The differences in the rates andthe shapes of the plots in FIG. 29 illustrates the need to monitor thereactions at several points in time. No attempt was made to correlatethe reaction mechanism with the kinetics in this work.

[0200] In summary, a new methodology, nonaqueous capillary arrayelectrophoresis coupled with microreaction, is developed to address thethroughput needs of combinatorial approaches to homogeneous catalysisand reaction optimization. Catalytic activity, selectivity and kineticsof the various combinations are determined quickly. This method ispotentially useful in the screening for asymmetric catalysts and drugsand combinatorial library synthesis.

INCORPORATION BY REFERENCE

[0201] All sources (e.g., inventor's certificates, patent applications,patents, printed publications, repository accessions or records, utilitymodels, World-Wide Web pages, and the like) referred to or citedanywhere in this document or in any drawing, Sequence Listing, orStatement filed concurrently herewith are hereby incorporated into andmade part of this specification by such reference thereto.

GUIDE TO INTERPRETATION

[0202] The foregoing is an integrated description of the invention as awhole, not merely of any particular element or facet thereof. Thedescription describes “preferred embodiments” of this invention,including the best mode known to the inventors for carrying it out. Ofcourse, upon reading the foregoing description, variations of thosepreferred embodiments will become obvious to those of ordinary skill inthe art. The inventors expect ordinarily skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law.

[0203] As used in the foregoing description and in the following claims,singular indicators (e.g., “a” or “one”) include the plural, unlessotherwise indicated. Recitation of a range of discontinuous values isintended to serve as a shorthand method of referring individually toeach separate value falling within the range, and each separate value isincorporated into the specification as if it were individually listed.As regards the claims in particular, the term “consisting essentiallyof” indicates that unlisted ingredients or steps that do not materiallyaffect the basic and novel properties of the invention can be employedin addition to the specifically recited ingredients or steps. Incontrast, the terms “comprising,” “having,” or “incorporating” indicatethat any ingredients or steps can be present in addition to thoserecited. The term “consisting of” indicates that only the recitedingredients or steps are present, but does not foreclose the possibilitythat equivalents of the ingredients or steps can substitute for thosespecifically recited.

What is claimed is:
 1. A parallel capillary electrophoresis system forseparating and analyzing the components of multiple chemical samples,said system comprising a bundle of capillary tubes arrayed to have atleast portions of the tubes extending generally parallel to one anotherin a first plane, each tube being adapted for the flow of a fluid sampletherethrough, a power source for applying a potential difference betweeninlet end portions and outlet end portions of the tubes to cause anelectrical current to flow through the contents of the capillary tubesat a level sufficient to cause separation in said fluid samples, a lightsource for emitting light to pass through said capillary tube portions,a photodetector comprising a linear array of photodetector elements forreceiving light passing through said capillary tubes, the light passingthrough each of said capillary tube portions illuminating severalphotodetector elements, each said photodetector element generating apixel signal corresponding to the light received by said photodetectorelement, an analog to digital converter converting each of the pixelsignals into a digital value corresponding to the light received by oneof the photodetector elements, and a processor receiving the digitalvalues and generating a plurality of output signals correspondingthereto, each output signal being a function of at least two digitalvalues corresponding to the light received by two photodetectorelements, respectively, so that the output signals correspond to thelight passing through the bundle of capillary tubes; wherein theprocessor generates output signals such that each output signal is afunction of at least two digital values corresponding to the lightpassing substantially concurrently through two photodetector elements,respectively.
 2. The system as set forth in claim 1 wherein theprocessor selects one digital value and averages the selected digitalvalue with at least a second digital value to generate averaged valuesand wherein the output signals are a function of the averaged values. 3.The system as set forth in claim 2 wherein the selected digital valueand the second digital value correspond to pixel signals from contiguousphotodetector elements.
 4. The system as set forth in claim 1 whereineach pixel signal is converted into a sequence of digital values andwherein the processor provides output signals which are a function ofthe sequence of digital values.
 5. The system as set forth in claim 1wherein the at least two digital values are selected to minimize shorttime fluctuations or other noise of the pixel signals to generate animproved signal to noise ratio of the pixel signals.
 6. A parallelcapillary electrophoresis system for separating and analyzing thecomponents of multiple chemical samples, said system comprising a bundleof capillary tubes arrayed to have at least portions of the tubesextending generally parallel to one another in a first plane, each tubebeing adapted for the flow of a fluid sample therethrough, a powersource for applying a potential difference between inlet end portionsand outlet end portions of the tubes to cause an electrical current toflow through the contents of the capillary tubes at a level sufficientto cause separation in said fluid samples, a light source for emittinglight to pass through said capillary tube portions, a photodetectorcomprising a linear array of photodetector elements for receiving lightpassing through said capillary tubes, the light passing through eachsaid capillary tube portions illuminating several photodetectorelements, each said photodetector element generating a pixel signalcorresponding to the light received by said photodetector element, ananalog to digital converter converting each of the pixel signals into adigital value corresponding to the light received by one of thephotodetector elements, and a processor receiving the digital values andgenerating a plurality of output signals corresponding thereto, eachoutput signal being a function of at least two digital valuescorresponding to the light received by two photodetector elements,respectively, so that the output signals correspond to the light passingthrough the bundle of capillary tubes; wherein the processor selects onepeak digital value and averages the selected digital value with fourdigital values which correspond to pixel signals from photodetectorelements adjacent to the photodetector element corresponding to theselected peak digital value and wherein the output signals are afunction of the averaged values.
 7. The system as set forth in claim 6wherein the selected peak digital value corresponds to the light passingthrough one capillary tube portion.
 8. A parallel capillaryelectrophoresis system for separating and analyzing the components ofmultiple chemical samples, said system comprising a bundle of capillarytubes arrayed to have at least portions of the tubes extending generallyparallel to one another in a first plane, each tube being adapted forthe flow of a fluid sample therethrough, a power source for applying apotential difference between inlet end portions and outlet end portionsof the tubes to cause an electrical current to flow through the contentsof the capillary tubes at a level sufficient to cause separation in saidfluid samples, a light source for emitting light to pass through saidcapillary tube portions, a photodetector comprising a linear array ofphotodetector elements for receiving light passing through saidcapillary tubes, the light passing through each of said capillary tubeportions illuminating several photodetector elements, each of saidphotodetector elements generating a pixel signal corresponding to thelight received by said photodetector element, an analog to digitalconverter converting each of the pixel signals into a digital valuecorresponding to the light received by one of the photodetectorelements, and a processor receiving the digital values and generating aplurality of output signals corresponding thereto, each output signalbeing a function of at least two digital values corresponding to thelight received by two photodetector elements, respectively, so that theoutput signals correspond to the light passing through the bundle ofcapillary tubes; wherein the processor selects one peak digital valueand averages the selected digital value with at least a second digitalvalue to generate averaged values and wherein the output signals are afunction of the averaged values, and further comprising a displayreceiving the output signals and generating an electropherogramcorresponding thereto.
 9. A parallel capillary electrophoresis systemfor separating and analyzing the components of multiple chemicalsamples, said system comprising: a bundle of capillary tubes arrayed tohave at least portions of the tubes extending generally parallel to oneanother in a first plane, each tube being adapted for the flow of afluid sample therethrough; a power source for applying a potentialdifference between inlet end portions and outlet end portions of thetubes to cause an electrical current to flow through the contents of thecapillary tubes at a level sufficient to cause separation in said fluidsamples; a light source for emitting light to pass through saidcapillary tube portions; a photodetector comprising a linear array ofphotodetector elements for receiving light passing through saidcapillary tubes, said linear array being positioned non-parallel to thefirst plane, the light passing through each of said capillary tubeportions illuminating several photodetector elements, each saidphotodetector element generating a pixel signal corresponding to thelight received by said photodetector element; an analog to digitalconverter converting each of the pixel signals into a digital valuecorresponding to the light received by one of the photodetectorelements; and a processor receiving the digital values and generating aplurality of output signals corresponding thereto, each output signalbeing a function of at least two digital values corresponding to thelight received by two photodetector elements, respectively, so that theoutput signals correspond to the light passing through the bundle ofcapillary tubes; wherein each pixel signal is converted into a sequenceof digital values and the output signals are a function of an averageover time of the sequence of digital values.
 10. A parallel capillaryelectrophoresis system for separating and analyzing the components ofmultiple chemical samples, said system comprising a bundle of capillarytubes arrayed to have at least portions of the tubes extending generallyparallel to one another in a first plane, each tube being adapted forthe flow of a fluid sample therethrough, a power source for applying apotential difference between inlet end portions and outlet end portionsof the tubes to cause an electrical current to flow through the contentsof the capillary tubes at a level sufficient to cause separation in saidfluid samples, a light source for emitting light to pass through saidcapillary tube portions, a photodetector comprising a linear array ofphotodetector elements for receiving light passing through saidcapillary tubes, the light passing through each said capillary tubeportions illuminating several photodetector elements, each saidphotodetector element generating a pixel signal corresponding to thelight received by said photodetector element, an analog to digitalconverter converting each of the pixel signals into a digital valuecorresponding to the light received by one of the photodetectorelements, and a processor receiving the digital values and generating aplurality of output signals corresponding thereto, each output signalbeing a function of at least two digital values corresponding to thelight received by two photodetector elements, respectively, so that theoutput signals correspond to the light passing through the bundle ofcapillary tubes; wherein the at least two digital values are selected tominimize long time drifts of the pixel signals to generate asubstantially flat baseline of the pixel signals.
 11. A method ofprocessing a plurality of pixel signals, each generated by one elementof an array of photodetector elements illuminated by light passingthrough a bundle of capillary tubes during a multiplexed capillaryelectrophoresis process, said method comprising: converting each of thepixel element signals into a digital value corresponding to the lightreceived by one of the photodetector elements; selecting, for eachcapillary tube, at least two digital values corresponding to the lightreceived by two photodetector elements; and generating output signalscorresponding to the light passing through the bundle of capillarytubes, each output signal being a function of the selected digitalvalues, wherein each output signal is a function of at least two digitalvalues corresponding to the light passing substantially concurrentlythrough two photodetector elements, respectively.
 12. The method as setforth in claim 11 comprising selecting one digital value and averagingthe selected digital value with at least a second digital value togenerate averaged values and wherein the output signals are a functionof the averaged values.
 13. The method as set forth in claim 12 whereinthe selected digital value and the second digital value correspond topixel signals from contiguous photodetector elements.
 14. The method asset forth in claim 11 wherein each pixel signal is converted into asequence of digital values and wherein the output signals are a functionof the sequence of digital values.
 15. The method as set forth in claim11 wherein the at least two digital values are selected to minimizeshort time fluctuations or other noise of the pixel signals to generatean improved signal to noise ratio of the pixel signals.
 16. A method ofprocessing a plurality of pixel signals, each generated by one elementof an array of photodetector elements illuminated by light passingthrough a bundle of capillary tubes during a multiplexed capillaryelectrophoresis process, said method comprising: converting each of thepixel element signals into a digital value corresponding to the lightreceived by one of the photodetector elements; selecting, for eachcapillary tube, at least two digital values corresponding to the lightreceived by two photodetector elements; generating output signalscorresponding to the light passing through the bundle of capillarytubes, each output signal being a function of the selected digitalvalues; and selecting one peak digital value and averaging the selecteddigital value with four digital values, which correspond to pixelsignals from photodetector elements adjacent to the photodetectorelement corresponding to the selected peak digital value and wherein theoutput signals are a function of the averaged values.
 17. The method asset forth in claim 16 wherein the selected peak digital valuecorresponds to the light passing through one capillary tube portion. 18.A method of processing a plurality of pixel signals, each generated byone element of an array of photodetector elements illuminated by lightpassing through a bundle of capillary tubes during a multiplexedcapillary electrophoresis process, said method comprising: convertingeach of the pixel element signals into a digital value corresponding tothe light received by one of the photodetector elements; selecting, foreach capillary tube, at least two digital values corresponding to thelight received by two photodetector elements; generating output signalscorresponding to the light passing through the bundle of capillarytubes, each output signal being a function of the selected digitalvalues; and selecting one peak digital value and averaging the selecteddigital value with at least a second digital value to generate averagedvalues and wherein the output signals are a function of the averagedvalues, and further comprising displaying an electropherogramcorresponding to the output signals.
 19. A method of processing aplurality of pixel signals, each generated by one element of an array ofphotodetector elements illuminated by light passing through a bundle ofcapillary tubes during a multiplexed capillary electrophoresis process,said bundle of capillary tubes arrayed to have at least portions of thetubes extending generally parallel to one another in a first plane, saidmethod comprising: positioning the array of photodetector elementsnon-parallel to the first plane; converting each pixel signal into asequence of digital values; a digital value corresponding to the lightreceived by one of the photodetector elements; selecting, for eachcapillary tube, at least two digital values corresponding to the lightreceived by two photodetector elements; and generating output signalscorresponding to the light passing through the bundle of capillarytubes, wherein the output signals are a function of an average over timeof the sequence of digital values.
 20. A method of processing aplurality of pixel signals, each generated by one element of an array ofphotodetector elements illuminated by light passing through a bundle ofcapillary tubes during a multiplexed capillary electrophoresis process,said method comprising: converting each of the pixel element signalsinto a digital value corresponding to the light received by one of thephotodetector elements; selecting, for each capillary tube, at least twodigital values corresponding to the light received by two photodetectorelements, wherein the at least two digital values are selected tominimize long time drifts of the pixel signals to generate asubstantially flat baseline of the pixel signals; and generating outputsignals corresponding to the light passing through the bundle ofcapillary tubes, each output signal being a function of the selecteddigital values.
 21. A parallel capillary electrophoresis system forseparating and analyzing the components of multiple chemical samples,said system comprising: a bundle of capillary tubes arrayed to have atleast portions of the tubes extending generally parallel to one anotherin a first plane, each tube being adapted for the flow of a fluid sampletherethrough; a power source for applying a potential difference betweeninlet end portions and outlet end portions of the tubes to cause anelectrical current to flow through the contents of the capillary tubesat a level sufficient to cause separation in said fluid samples; a lightsource for emitting light to pass through said capillary tube portions;a photodetector comprising a linear array of photodetector elements forreceiving light passing through said capillary tubes, said linear arraybeing positioned non-parallel to the first plane, the light passingthrough each of said capillary tube portions illuminating severalphotodetector elements, each of said photodetector element generating apixel signal corresponding to the light received by said photodetectorelement; an analog to digital converter converting each of the pixelsignals into a digital value corresponding to the light received by oneof the photodetector elements; and a processor receiving the digitalvalues and generating a plurality of output signals correspondingthereto, each output signal being a function of at least two digitalvalues corresponding to the light received by two photodetectorelements, respectively, so that the output signals correspond to thelight passing through the bundle of capillary tubes.
 22. A system as setforth in claim 21 wherein the linear array is positioned generallyperpendicular to the first plane.
 23. A system as set forth in claim 21wherein the processor generates output signals such that each outputsignal is a function of at least two digital values corresponding to thelight passing substantially concurrently through two photodetectorelements, respectively.
 24. A system for use in analyzing multiplesamples simultaneously by absorption detection, which system comprises:(i) a planar array of multiple containers, into each of which can beplaced a sample, (ii) a light source for emitting light to pass throughthe planar array of multiple containers, (iii) a photodetector, which isin line with the light source, is positioned in line with and parallelto the planar array of multiple containers, and comprises a linear arrayof photosensitive elements for receiving light passing through theplanar array of multiple containers, wherein, upon illumination of aphotosensitive element by light passing through the planar array ofmultiple containers, a pixel signal corresponding to the light receivedby the photosensitive element is generated, (iv) an analog to digitalconverter, which converts the pixel signal for each illuminatedphotosensitive element to a digital value corresponding to the lightreceived by the respective photosensitive element, and (v) a processor,which receives the digital values and generates a plurality of outputsignals corresponding thereto, each output signal being a function of atleast two digital values corresponding to the light passingsubstantially concurrently through two photosensitive elements.
 25. Thesystem of claim 24, wherein the processor selects one peak digital valueand averages the selected digital value with four digital values, whichcorrespond to pixel signals from photosensitive elements adjacent to thephotosensitive element corresponding to the selected peak digital value,and wherein the output signals are a function of the averaged values.26. The system of claim 24, wherein the processor selects one peakdigital value and averages the selected digital value with at least asecond digital value to generate averaged values and wherein the outputsignals are a function of the averaged values, and further comprising adisplay receiving the output signals and generating an electropherogramcorresponding thereto.
 27. The system of claim 24, wherein each pixelsignal is converted into a sequence of digital values and the outputsignals are a function of an average over time of the sequence ofdigital values.
 28. The system of claim 24, wherein the at least twodigital values are selected to minimize long time drifts of the pixelsignals to generate a substantially flat baseline of the pixel signals.29. A method of processing a plurality of pixel signals, each generatedby one element of an array of photosensitive elements illuminated bylight passing through a planar array of multiple containers, said methodcomprising: converting each of the pixel element signals into a digitalvalue corresponding to the light received by one of the photosensitiveelements; selecting, for each container, at least two digital valuescorresponding to the light received by two photosensitive elements; andgenerating output signals corresponding to the light passing through theplanar array of multiple containers, each output signal being a functionof the selected digital values, wherein each output signal is a functionof at least two digital values corresponding to the light passingsubstantially concurrently through two photosensitive elements,respectively.
 30. A method of processing a plurality of pixel signals,each generated by one element of an array of photosensitive elementsilluminated by light passing through a planar array of multiplecontainers, said method comprising: converting each of the pixel elementsignals into a digital value corresponding to the light received by oneof the photosensitive elements; selecting, for each container, at leasttwo digital values corresponding to the light received by twophotosensitive elements; generating output signals corresponding to thelight passing through the planar array of multiple containers, eachoutput signal being a function of the selected digital values; andselecting one peak digital value and averaging the selected digitalvalue with four digital values, which correspond to pixel signals fromphotosensitive elements adjacent to the photosensitive elementcorresponding to the selected peak digital value and wherein the outputsignals are a function of the averaged values.
 31. A method ofprocessing a plurality of pixel signals, each generated by one elementof an array of photosensitive elements illuminated by light passingthrough a planar array of multiple containers, said method comprising:converting each of the pixel element signals into a digital valuecorresponding to the light received by one of the photosensitiveelements; selecting, for each container, at least two digital valuescorresponding to the light received by two photosensitive elements;generating output signals corresponding to the light passing through theplanar array of multiple containers, each output signal being a functionof the selected digital values; and selecting one peak digital value andaveraging the selected digital value with at least a second digitalvalue to generate averaged values and wherein the output signals are afunction of the averaged values, and further comprising displaying anelectropherogram corresponding to the output signals.
 32. A method ofprocessing a plurality of pixel signals, each generated by one elementof an array of photosensitive elements illuminated by light passingthrough a planar array of multiple containers, said planar array ofmultiple containers arrayed to have at least portions of the containersextending generally parallel to one another in a first plane, saidmethod comprising: positioning the array of photosensitive elementsnon-parallel to the first plane; converting each pixel signal into asequence of digital values; a digital value corresponding to the lightreceived by one of the photosensitive elements; selecting, for eachcontainer, at least two digital values corresponding to the lightreceived by two photosensitive elements; and generating output signalscorresponding to the light passing through the planar array of multiplecontainers, wherein the output signals are a function of an average overtime of the sequence of digital values.
 33. A method of processing aplurality of pixel signals, each generated by one element of an array ofphotosensitive elements illuminated by light passing through a planararray of multiple containers, said method comprising: converting each ofthe pixel element signals into a digital value corresponding to thelight received by one of the photosensitive elements; selecting, foreach container, at least two digital values corresponding to the lightreceived by two photosensitive elements, wherein the at least twodigital values are selected to minimize long time drifts of the pixelsignals to generate a substantially flat baseline of the pixel signals;and generating output signals corresponding to the light passing throughthe planar array of multiple containers, each output signal being afunction of the selected digital values.
 34. A system for use inanalyzing multiple samples simultaneously by absorption detection, whichsystem comprises: (i) a planar array of multiple containers, into eachof which can be placed a sample, (ii) a light source for emitting lightto pass through the planar array of multiple containers, (iii) aphotodetector, which is in line with the light source, is positioned inline with and parallel to the planar array of multiple containers, andcomprises a linear array of photosensitive elements for receiving lightpassing through the planar array of multiple containers, wherein, uponillumination of a photosensitive element by light passing through theplanar array of multiple containers, a pixel signal corresponding to thelight received by the photosensitive element is generated, (iv) ananalog to digital converter, which converts the pixel signal for eachilluminated photosensitive element to a digital value corresponding tothe light received by the respective photosensitive element, and (v) aprocessor, which receives the digital values and generates a pluralityof output signals corresponding thereto, each output signal being afunction of at least two digital values corresponding to the lightreceived by two photosensitive elements, respectively, so that theoutput signals correspond to the light passing through the planar arrayof multiple containers.